ES2684606T3 - Detergent composition - Google Patents

Abstract

Use of a polypeptide that has DNase activity to prevent, reduce or eliminate a biofilm of an article, where the polypeptide is obtained from a fungal source and the article is a textile and where the polypeptide that has DNase activity has at least 80% sequence identity with the mature polypeptide of SEQ ID NO: 9.

Description

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DESCRIPTION

Detergent composition Reference to a sequence listing

[0001] This application contains a list of sequences in computer readable format, which is incorporated herein by reference.

Field of the Invention

[0002] The present invention relates to a detergent and pharmaceutical composition comprising a deoxyribonuclease (DNase), wherein DNase is obtained from a fungal source. In addition, it refers to a washing method and the use of DNase. The present invention further relates to polypeptides having DNase activity, nucleotides encoding the polypeptide, as well as methods of producing the polypeptides.

Background of the invention

[0003] Microorganisms generally live fixed to surfaces in many natural, industrial and medical environments, encapsulated by extracellular substances that include biopolymers and macromolecules. The resulting layer of encapsulated microorganism silt is called a biofilm. Biofilms are the predominant mode of growth of bacteria in the natural environment, and bacteria that grow in biofilms exhibit different physiological properties. Compared to their equivalents that have grown planktonically, the bacteria in a biofilm are more resistant to antibiotics, UV irradiation, detergents and the host's immune response.

[0004] A biofilm may include one or more microorganisms, including gram-positive and gram-negative bacteria, algae, protozoa, and / or yeast or filamentous fungi and viruses and / or bacteriophages. Examples of problematic biofilms are dental plaque, infections in medical implants, but also initial contamination in ship hulls. Biofilms are attributed to the pathogenesis of many infections in humans and are a significant problem in the industry in terms of biocontamination of exposed surfaces, where biofilm colonization can form the base component of a localized ecosystem that can interrupt and interfere with industrial processes and components.

[0005] When laundry items such as shirts or sportswear are used, they are exposed to bacteria from the user's body and the rest of the environment in which they are used. Some of these bacteria are able to adhere to the laundry article and form a biofilm in the article. The presence of bacteria implies that laundry items become sticky and, therefore, dirt adheres to sticky areas. This dirt has proved difficult to remove by commercially available detergent compositions. In addition, when very dirty laundry items are washed together with less dirty laundry items, the dirt present in the wash solution tends to stick to the biofilm. As a result, the laundry article is more "stained" after washing than before washing. In addition, these bacteria are a source of bad smell, which develops after the use of the laundry article. The bad smell is difficult to remove and can remain even after washing. The reason for this bad smell is the adhesion of bacteria to the surface of the textile. Due to adhesion to the textile, bacteria can remain even after washing and continue to be a source of bad smell.

[0006] International patent application WO 2011/098579 refers to bacterial deoxyribonuclease compounds and methods for interrupting and preventing biofilm.

Summary of the Invention

[0007] The present invention relates to a detergent composition comprising a polypeptide having deoxyribonuclease activity (DNase) and a detergent adjunct ingredient, where DNase is obtained from a fungal source and where the polypeptide is obtained from Aspergillus. The invention further relates to the use of a polypeptide having DNase activity to prevent, reduce or eliminate a biofilm from an article, where the polypeptide is obtained from a fungal source and the article is a textile and where the polypeptide having DNase activity has at least 80% sequence identity with the mature polypeptide of SEQ iD #: 9.

[0008] The invention also relates to a method of washing an article that includes the steps of:

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to. Exposing an article to a wash solution comprising a polypeptide where the polypeptide having DNase activity has at least 80% sequence identity with the mature polypeptide of SEQ ID No. 9 or a detergent composition according to any of claims 8-10;

b. Complete at least one wash cycle; Y

C. Optionally rinse the article, where the article is a textile.

Definitions

[0009] Allelic variant: the term "allelic variant" means any of two or more alternative forms of a gene that occupies the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in polymorphism within populations. Genetic mutations can be silent (without change in the encoded polypeptide) or they can encode polypeptides that have altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0010] Biofilm: A biofilm is any group of microorganisms in which cells stick together on a surface, such as a textile, crockery or hard surface. These adherent cells are frequently introduced into a self-produced matrix of extracellular polymeric substance (EPS). The EPS of the biofilm is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms can be formed on living or non-living surfaces. Microbial cells that grow in a biofilm are physiologically different from planktonic cells of the same organism, which, in contrast, are unique cells that can float or swim in a liquid medium.

[0011] Bacteria that live in a biofilm usually have significantly different properties from bacteria that float freely from the same species, since the dense and protected environment of the film allows them to cooperate and interact in several ways. A benefit of this environment is the increased resistance to detergents and antibiotics, since the dense extracellular matrix and the outer layer of cells protect the interior of the community.

[0012] In laundry, bacteria that produce biofilms can be found among the following species: Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp.

[0013] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a spliced mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. The cDNA lacks intron sequences that may be present in the corresponding genomic DNA. Primary initial RNA transcription is a precursor for mRNA that is processed through a series of stages, including splicing, before appearing as mature spliced mRNA.

[0014] Coding sequence: the term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The limits of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with an end codon such as TAA, TAG or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0015] Color difference (L value): a Lab color space is a space of opposite colors with an L dimension for brightness. Value L, L * represents the darkest black in L * = 0, and the brightest white in L * = 100. In the context of the present invention, the value L is also referred to as a color difference.

[0016] Control sequences: the term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence can be native (i.e., of the same gene) or foreign (i.e., of a different gene) to the polynucleotide encoding the polypeptide or native or foreign between them. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences can be provided with linkages in order to introduce specific restriction sites that facilitate ligament of control sequences with the polynucleotide coding region encoding a polypeptide.

[0017] The term "deep cleaning" means interruption or elimination of a biofilm or components of a biofilm such as polysaccharides, proteins, DNA, dirt or other components present in the biofilm.

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[0018] Detergent attached ingredient: The detergent attached ingredient is different from the DNase of this invention. The precise nature of these additional attached components, and the levels of incorporation thereof, will depend on the physical form of the composition and the nature of the operation for which it will be used. Suitable adjunctive materials include, but are not limited to, the components described below such as surfactants, adjuvants, flocculation aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators , hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay dirt removal / antiredeposition agents, brighteners, soapy suppressants, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers , hydrotropes, adjuvants and adjuvants, tissue matting agents, antifoaming agents, dispersants, processing aids, and / or pigments.

[0019] Detergent composition: the term "detergent composition" refers to compositions that find their use in the removal of unwanted compounds from items to be cleaned, such as fabrics. The detergent composition can be used, for example, to clean textiles for both home cleaning and industrial cleaning. The terms encompass any materials / compounds selected for the particular type of cleaning composition desired and the form of the product (for example, liquid, gel, powder, granulate, paste, or powder compositions) and include, but not limiting, detergent compositions (eg, liquid and / or solid laundry detergents and mild fabric detergents; fabric fresheners; fabric softeners; pre-identifiers / pretreatment of laundry textiles). In addition to containing the enzyme of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mananas , or any mixture thereof), and / or attached detergent ingredients such as surfactants, adjuvants, chelants or chelating agents, bleaching system or bleaching components, polymers, fabric conditioners, foam enhancers, soap suppressants, dyes, perfume, tanning inhibitors, optical brighteners, bactericides, fungicides, soil suspension agents, anticorrosive agents, enzyme inhibitors or stabilizers, enzymatic activators, transferase (s), hydrolytic enzymes, oxidoreductases, bluish agents and fluorescent dyes, antioxidants, and solubilizers.

[0020] DNase (deoxyribonuclease): the term "DNase" means a polypeptide with DNase activity that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA structure, thereby degrading the DNA. For the purposes of the present invention, DNase activity is determined according to the procedure described in test I. In one aspect, the polypeptides of the present invention have at least 20%, for example, at least 40%, at minus 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the DNase activity of the mature polypeptide of SEQ ID NO: 2. In one embodiment, the polypeptides of the present invention have enhanced DNase activity, for example such that the DNase activity of the polypeptide is at least 105%, for example, at minus 110%, at least 120%, at least 130%, at least 140%, at least 160%, at least 170%, at least 180%, or at least 200% with reference to DNase activity of the mature polypeptide of SEQ ID NO: 2.

[0021] Benefit of enzymatic detergency: the term "benefit of enzymatic detergency" is defined herein as the advantageous effect that an enzyme can add to a detergent compared to the same detergent without the enzyme. Important detergency benefits that can be provided by enzymes are the removal of stains without any dirt or with very little visible dirt after washing and / or cleaning, prevention or reduction of the redeposition of the dirt released in the process washing (an effect that is also called anti-redeposition), the complete or partial restoration of the whiteness of textiles that were originally white, but that after use and repeated washing have obtained a grayish or yellowish appearance (an effect that is also called whitening). The benefits of textile care, which are not directly related to the removal of catalytic stains or the prevention of dirt redeposition, are also important for the benefits of enzymatic detergency. Examples of such textile care benefits are the prevention or reduction of the transfer of dyes from one fabric to another fabric or another part of the same fabric (an effect that is also called inhibition of dye transfer or anti-restraint), elimination of broken or protruding fibers from a surface of the fabric to reduce fading tendencies or eliminate existing pellets or lint (an effect that is also called antifrising), improvement of tissue softness, tissue color lightening and the removal of particulate dirt that is retained in the fibers of the fabric or garment. Enzymatic bleaching is a benefit of additional enzymatic detergency where catalytic activity is generally used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.

[0022] Expression: The term "expression" includes any step involved in the production of a polypeptide that includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

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[0023] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide that encodes a polypeptide and is operably linked to control sequences that provide its expression.

[0024] Fragment: the term "fragment" means a polypeptide having one or more (eg, several) amino acids absent from the amino and / or carboxyl terminal of a mature polypeptide or domain; where the fragment has DNase activity. In one aspect, a fragment contains at least 206 amino acid residues (for example, amino acids 1 to 206 of SEQ ID NO: 2), at least 205 amino acid residues (for example, amino acids 2 to 206 of the SEQ ID No.: 2), or at least 204 amino acid residues (for example, amino acids 3 to 206 of SEQ ID No. 2).

[0025] Fungal: in the context of the present invention, the term "fungal" in relation to a polypeptide (such as an enzyme, for example, a DNase) refers to a polypeptide encoded by, and therefore directly derivable from, the genome of a fungus, where such fungus has not been genetically modified to encode said polypeptide, for example, by introducing the coding sequence into the genome by recombinant DNA technology. In the context of the present invention, the term "fungal DNase" or "polypeptide having DNase activity obtained from a fungal source" refers, therefore, to a DNase encoded by, and therefore directly derivable from genome of a fungal species, where the fungal species has not undergone a genetic modification that introduces recombinant DNA encoding said DNase. Thus, the nucleotide sequence encoding the fungal polypeptide that has DNase activity is a natural sequence in the genetic context of a fungal species. The fungal polypeptide that has DNase activity coding by such a sequence can also refer to a wild-type DNase (or progenitor DNase). In another aspect, the invention provides polypeptides having DNase activity, wherein said polypeptides are substantially homologous to a fungal DNase. In the context of the present invention, the term "substantially homologous" denotes a polypeptide having DNase activity that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98% and most preferably at least 99% identical to the amino acid sequence of a selected fungal DNase. Polypeptides that are substantially homologous to a fungal DNase can be included in the detergent of the present invention and / or used in the methods of the present invention.

[0026] Host cell: the term "host cell" means any type of cell that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a progenitor cell that is not identical to the progenitor cell due to mutations that occur during replication.

[0027] Improved wash performance: the term "improved wash performance" is defined herein as an enzyme that shows higher wash performance in a detergent composition with respect to the wash performance of the same detergent composition without the enzyme, for example, with greater stain removal or less redeposition. The term "improved wash performance" includes wash performance in laundry.

[0028] Isolated: the term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any substance that does not occur naturally, (2) any substance, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, which is eliminated at least partially of one or more or all of the constituents that occur naturally with those associated in nature; (3) any substance modified by the hand of man with respect to that substance found in nature; or (4) any substance modified by increasing the amount of the substance with respect to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a sample of fermentation broth; for example, a host cell can be genetically modified to express the polypeptide of the invention. The fermentation broth of that host cell will comprise the isolated polypeptide.

[0029] Washing: the term "washing" refers to both home washing and industrial washing and means the process of treating textiles with a solution containing a cleaning or detergent composition of the present invention. The washing process can be carried out, for example, using a domestic or industrial washing machine or it can be carried out by hand.

[0030] The term "bad smell" means a smell that is not desired in clean items. The cleaned item should smell fresh and clean without bad odors attached to the item. An example of a bad smell is a compound with a smell

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unpleasant, which may be produced by microorganisms. Another example of unpleasant odors may be the smell of sweat or body odor attached to an article that has been in contact with a human or animal. Another example of a bad smell may be the smell of spices, which sticks to items, for example, curry or other exotic spices that smell strongly. One way to measure the ability of an article to adhere bad smell is the use of test II described in this document.

[0031] Mature polypeptide: the term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modification, such as N-terminal treatment, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 206 of SEQ ID No. 2 and amino acids -37 to -16 of SEQ ID No. 2 are a signal peptide and amino acids -15 to - 1 of SEQ ID No.: 2 are a propeptide. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently and, therefore, a host cell that expresses from a polynucleotide can produce a different mature polypeptide (for example, with a different C-terminal and / or N-terminal amino acid ) compared to another host cell that expresses the same polynucleotide. In one aspect, a mature polypeptide contains up to 206 amino acid residues and of SEQ ID NO: 2 or SEQ ID NO: 9 (for example, amino acids 1 to 206 of SEQ ID NO: 2 ), or up to 204 amino acid residues (for example, amino acids 3 to 206 of SEQ ID NO: 2).

[0032] Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having DNase activity. In one aspect, the coding sequence of the mature polypeptide is nucleotides linked 1 to 242, 309 to 494, 556 to 714 and 766 to 907 of SEQ ID NO: 1.

[0033] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single or double stranded, that is isolated from a gene that occurs naturally or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or that is synthetic, comprising one or more control sequences.

[0034] Operationally linked: the term "operably linked" means a configuration in which a control sequence is placed in an appropriate position with respect to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence

[0035] Pharmaceutical attached ingredient means any pharmaceutical excipient suitable for the formulation of the pharmaceutical compound.

[0036] Such excipients, carriers, vehicles, etc. they are well known to those skilled in the art and are described in textbooks such as Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985.

[0037] Pharmaceutically acceptable excipients that are suitable for use in tablet formulations include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a prolonged action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be used.

[0038] For hard gelatin capsule formulations, the active substance may be mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. For soft gelatin capsule formulations, the active substance can be mixed with water or an oily medium, for example, peanut oil, liquid paraffin or olive oil.

[0039] Suitable excipients for the production of aqueous suspensions include suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and acacia gum; dispersing agents or humectants may be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic chain alcohols long, for example heptadecaethylene oxyethanol, or condensation products of ethylene oxide with partial esters obtained from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or ethylene oxide condensation products

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with partial esters obtained from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate.

[0040] Aqueous suspensions may also contain one or more preservatives, for example, benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents. , such as sucrose or saccharin.

[0041] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example, peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

[0042] Remission value: the washing performance is expressed as a remission value of the stained samples. After washing and rinsing, the samples were spread flat and allowed to air dry at room temperature overnight. All washed samples were evaluated the day after washing. Sample light reflectance evaluations were made using a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. The measurements were made without UV in the incident light and the remission was extracted at 460 nm.

[0043] Sequence identity: the relationship between two amino acid sequences or between two nucleotide sequences is described by the "sequence identity" parameter. For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program from the EMBOSS package (eMbOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276277), preferably version 5.0.0 or later. The parameters used are penalty for opening of space of 10, penalty for extension of space of 0.5, and the replacement matrix EBLOSUM62 (EMBOSS version of BLOSUM62). The result of Needle labeled "greater identity" (obtained using the option not summarized) is used as the percentage identity and is calculated as follows:

(Identical waste x 100) / (Alignment length - Total number of spaces in the alignment)

[0044] For the purposes of the present invention, sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS : The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are a space opening penalty of 10, a space extension penalty of 0.5, and the EDNAFULL replacement matrix (NCBI NUC4.4 EMBOSS version). The result of Needle labeled "greater identity" (obtained using the option not summarized) is used as the percentage identity and is calculated as follows:

(Identical deoxyribonucleotides x 100) / (Alignment length - Total number of spaces in the alignment)

[0045] Textile: the term "textile" means any textile material, including threads, thread intermediaries, fibers, nonwovens, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made of fabrics (for example, garments and other items). The textile or fabric can be in the form of stitches, fabrics, jeans, nonwovens, felts, threads and cloths. The textile may have a cellulose base such as natural cellulosics, including cotton, linseed / flax, jute, ramie, sisal or coconut cellulose or artificial cellulose (for example, originating from wood pulp), which include viscose / rayon, cellulose acetate (tricell) fibers, lyocell or mixture thereof. The textile or fabric may also have a non-cellulosic base, such as natural polyamides, including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex / elastane, or mixtures thereof as well as mixtures of fibers with cellulose base and non-cellulosic base. Examples of mixtures are cotton and / or rayon / viscose blends with one or more accompanying materials such as wool, synthetic fiber (for example, polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber , polyurea fiber, aramid fiber), and / or cellulose-containing fiber (for example, rayon / viscose, ramie, flaxseed / linen, jute, cellulose acetate fiber, lyocell). The fabric can be conventional washable laundry, for example, household stained laundry. When the term fabric or garment is used, it is also intended to include the broader term textiles. In the context of the present invention, the term "textile" also encompasses fabrics.

[0046] Astringency conditions: the term "very low astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200

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micrograms / ml of sperm DNA cut and denatured salmon DNA, and 25% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45 ° C.

[0047] The term "low astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / ml salmon sperm DNA cut and denatured, and 25% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50 ° C.

[0048] The term "medium astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / ml salmon sperm DNA cut and denatured, and 35% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55 ° C.

[0049] The term "medium-high astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / ml sperm DNA of cut and denatured salmon, and 35% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60 ° C.

[0050] The term "high astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / ml salmon sperm DNA cut and denatured, and 50% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 ° C.

[0051] The term "very high astringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sperm DNA of cut and denatured salmon, and 50% formamide, after standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70 ° C.

[0052] Subsequence: the term "subsequence" means a polynucleotide having one or more (eg, several) nucleotides absent from the 5 'and / or 3' end of a mature polypeptide coding sequence; where the sub sequence encodes a fragment that has DNase activity. In one aspect, a sub-sequence contains at least 796 nucleotides (for example, nucleotides 112 to 907 of SEQ ID No. 1), at least 793 nucleotides (for example, nucleotides 115 to 907 of SEQ ID N. °: 1), or at least 790 nucleotides (for example, nucleotides 118 to 907 of SEQ ID No.: 1).

[0053] Textile: the term "textile" means any textile material, including threads, thread intermediaries, fibers, nonwovens, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made of fabrics (for example, garments and other items). The textile or fabric can be in the form of dots, fabrics, jeans, nonwovens, felts, threads and cloths. The textile may have a cellulose base such as natural cellulosics, including cotton, linseed / linen, jute, ramie, sisal or coir or artificial cellulosics (for example, originating from wood pulp), including viscose / rayon, fibers of cellulose acetate (tricell), lyocell or mixture thereof. The textile or fabric can also have a non-cellulosic base such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acryl, polypropylene and spandex / elastane, or mixture of the same as mixtures of fibers with cellulose base and non-cellulosic base. Examples of mixtures are cotton and / or rayon / viscose blends with one or more accompanying materials such as wool, synthetic fiber (for example, polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber , polyurea fiber, aramid fiber), and / or cellulose-containing fiber (for example, rayon / viscose, ramie, flaxseed / linen, jute, cellulose acetate fiber, lyocell). The fabric can be conventional washable dirty laundry, for example, household stained laundry. When the term fabric or garment is used, it is also intended to include the broader term textiles. In the context of the present invention, the term "textile" also includes fabrics.

[0054] Variant: the term "variant" means a polypeptide that has the same activity as the parent enzyme that comprises an alteration, that is, a substitution, insertion, and / or elimination, in one or more (for example, several) positions. A substitution means an amino acid replacement that occupies a position with an amino acid

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different; a deletion means a suppression of the amino acid that occupies a position; and an insertion means an addition of an adjacent amino acid and immediately after the amino acid that occupies a position. In the context of the present invention, a variant of an identified DNase has the enzymatic activity of the parent, that is, the ability to catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA structure (deoxyribonuclease activity). In one embodiment, the deoxyribonuclease activity of the variant is increased with reference to the progenitor DNase, for example, the mature polypeptide of SEQ ID NO: 2.

[0055] Washing cycle: the term "washing cycle" is defined herein as a washing operation where textiles are immersed in the washing solution, mechanical action of some kind is applied to the textile to release stains and to facilitate the flow of the wash solution into and out of the textile and finally the excess wash solution is removed. After one or more wash cycles, the textile is usually rinsed and dried.

[0056] Washing solution: the term "washing solution" is defined herein as the solution or mixture of water and detergent components that optionally includes the enzyme of the invention.

[0057] Washing time: the term "washing time" is defined herein as the duration of a complete washing process; that is, the cycle time (the cycles) of washing and the cycle (the cycles) of rinsing together.

[0058] Whiteness: The term "whiteness" is defined in this document as a broad term with different meanings in different regions and for different consumers. The loss of whiteness may be due, for example, to graying, yellowing, or the removal of optical brighteners / shading agents. Grating and yellowing may be due to the redeposition of dirt, body dirt, coloration from, for example, iron and copper ions or the transfer of dyes. Whiteness may include one or more problems from the following list: effects of dye or dye; Incomplete stain removal (for example, body dirt, sebum, etc.); redeposition (graying, yellowing or other discolorations of the object) (the dirt removed is re-associated with other parts of the textile, dirty or not); chemical changes in the textile during application; and lightening or brightening of colors.

Detailed description of the invention

[0059] The inventors have discovered that polypeptides that have deoxyribonuclease (DNase) activity and that are obtained from a fungal source can be used to prevent or eliminate biofilm in articles such as textiles and / or tissue. The biofilm can develop in the textile when microorganisms are present in an article and stick to the article. Some microorganisms tend to adhere to the surface of items such as textiles. Some microorganisms adhere to such surfaces and form a biofilm on the surface. The biofilm can be sticky and the attached microorganisms and / or the biofilm can be difficult to remove. In addition, the biofilm adheres to dirt due to the sticky nature of the biofilm. Commercial laundry detergent compositions available in the label do not remove such adherent microorganisms or biofilm.

[0060] The present invention relates to the use of a polypeptide having DNase activity to prevent, reduce or eliminate a biofilm of an article, where the polypeptide having DNase activity is obtained from a fungal source and where the article It is a textile. In an embodiment of the invention, the polypeptide having DNase activity is used to prevent, reduce or eliminate the stickiness of an article. The polypeptide having DNase activity can also be used to pretreat textile stains such as a textile with a pronounced amount of biofilm adhered to the textile.

[0061] Additionally, the invention relates to the use of a polypeptide having DNase activity to prevent, reduce or eliminate redeposition of dirt during a wash cycle. When the polypeptide is used, for example, in the washing of a textile, the polypeptide hinders the deposition of the dirt present in the washing solution to be deposited in the textile.

[0062] Furthermore, the invention relates to the use of a polypeptide having DNase activity to prevent, reduce or eliminate adhesion of dirt to an article. In one embodiment, the article is textile. When the dirt does not adhere to the article, the article seems cleaner. Thus, the invention further relates to the use of a polypeptide having DNase activity to maintain or improve the whiteness of the article.

[0063] When items such as shirts or sportswear are used, they are exposed to bacteria from the user's body and the rest of the environment where they are worn. This can cause a bad smell in the item even after the item is washed. The present invention, therefore, also relates to the elimination or reduction of bad smell in the textile. The bad smell can be caused by bacteria compounds that produce compounds with an unpleasant smell. A

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An example of such unpleasant odor compounds is E-2-nonenal. The bad smell may be present in a freshly washed textile that is still wet. Or the bad smell may be present in a freshly washed textile, which has subsequently dried. The bad smell may also be present in a textile that has been stored for some time after washing. The present invention relates to the reduction or elimination of bad smell such as E-2- nonenal of wet or dry textile.

[0064] The present invention further relates to a detergent composition comprising a polypeptide having DNase activity and a detergent adjunct ingredient, where DNase is obtained from a fungal source. The present detergent composition can be used to prevent, reduce or eliminate the biofilm of an article, to prevent, reduce or eliminate the stickiness of an article, to pre-treat stains on the article, to prevent, reduce or eliminate the redeposition of dirt. during a wash cycle, to reduce or eliminate the adhesion of dirt to an article, to maintain or improve the whiteness of an article and to prevent, reduce or eliminate the bad smell of an article, such as E-2-nonenal ( see example 10, 13 and 14). The present detergent composition overcomes the problems of the prior art.

[0065] The inventors have surprisingly discovered that polypeptides having DNase activity obtained from a fungal source are more stable than bacterial polypeptides that have DNase activity when formulated with other detergent enzymes. Especially, the inventors have discovered that in formulating the DNase obtained from a fungal source in a composition comprising protease, the fungal DNase is more stable than the bacterial DNase obtained from a bacterial source. The polypeptide having DNase activity can be obtained from Aspergillus, for example, from Aspergillus oryzae. The polypeptide having DNase activity obtained from Aspergillus oryzae has been shown to be more stable when formulated with protease than the combination of bacterial DNase and protease (see example 11 and 12).

[0066] In one embodiment of the invention, the detergent composition comprises the polypeptide having DNase activity as claimed herein.

[0067] In one embodiment of the invention, the detergent attached ingredient is selected from the group consisting of surfactants, adjuvants, flocculation aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, materials catalytic, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay dirt removal / antiredeposition agents, brighteners, soaps suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, adjuvants and adjuvants, tissue matting agents, anti-foaming agents, dispersants, processing aids, and / or pigments.

[0068] The detergent attached ingredient may be a surfactant. An advantage of including a surfactant in a detergent composition comprising a fungal DNase is that washing performance is improved. In one embodiment, the detergent appended ingredient is an adjuvant or a clay dirt removal / antiredeposition agent.

[0069] In one embodiment, the detergent attached ingredient is an enzyme. The detergent composition may comprise one or more enzymes. The one or more enzymes can be selected from the group consisting of proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannases, arabinases, galactanases, xylanases and oxidases.

[0070] The inventors have discovered that DNase obtained from a fungal source shows good stability when formulated with other enzymes. DNase obtained from a bacterial source has been shown to have poor stability when bacterial DNase is formulated with other enzymes such as proteases. The inventors have discovered that DNase obtained from a fungal source has increased stability compared to DNase obtained from a bacterial source.

[0071] In one embodiment of the invention, the inventive detergent composition is formulated with a protease, which is of animal, plant or microbial origin. Protease is a chemically designed or modified protein. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.

[0072] In one embodiment of the invention, the protease is selected from the group consisting of Bacillus, for example, Novo subtilisin, Carlsberg subtilisin, 309 subtilisin, 147 subtilisin, 168 subtilisin 168, trypsin of bovine origin, trypsin of porcine origin and Fusarium protease. The protease may have at least 90%, such as at least 95%, of sequence identity with SEQ ID NO: 10. In one embodiment, the protease has at

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minus 90% identity with the amino acid sequence of SEQ ID #: 10 or a variant thereof with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274, preferably the variant is an alkaline protease that has at least 90% identity with the amino acid sequence of SEQ ID No .: 10 with the following replacement: M222S or the N76D + G195E substitutions.

[0073] In one embodiment, the detergent composition is capable of reducing the adhesion of bacteria selected from the group consisting of Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp. to a surface, or release bacteria from a surface to which they adhere.

[0074] In one embodiment of the invention, the surface is a textile surface. The textile can be made of cotton, cotton / polyester, polyester, polyamide, polyacryl and / or silk.

[0075] The detergent composition may be formulated as a tablet, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. The detergent composition may be a liquid detergent, a powder detergent or a granule detergent.

[0076] The invention further relates to a liquid detergent composition comprising a surfactant and a detergent adjuvant in a total concentration of at least 3% by weight, and a microcapsule containing detergent enzyme, where the microcapsule membrane is produced by crosslinking of a poly-branched polyamine having a molecular weight greater than 1 kDa. The inventors have discovered that by encapsulating enzymes in a microcapsule with a semipermeable membrane of the invention, and with a water activity inside these capsules (before the addition to the liquid detergent) than in the liquid detergent, the capsules will experience a collapse (partial) when added to the detergent (water oozes), thus leaving an interior that contains more concentrated and more viscous enzymes in the capsules. Collapse of the membrane can also result in reduced permeability. This can also be used by adding stabilizers / polymers, especially ones that are not permeable through the membrane. Collapse and the resulting increase in viscosity will reduce / hinder the diffusion of hostile components (e.g., surfactants or sequestrants) in the capsules, and thus increase the storage stability of the enzyme in the liquid detergent. Components in the liquid detergent that are sensitive to the enzyme (for example, the components that make substrate for the enzyme) are also protected against degradation by the enzyme. During washing, the liquid detergent is diluted with water, thus increasing water activity. Water will now diffuse in the capsules (osmosis). The capsules will swell and the membrane will become either permeable to the enzyme so that it can leave the capsules, or else it will simply burst and thus release the enzyme. The concept is very effective in stabilizing enzymes against hostile components in the liquid detergent, and vice versa, it also protects the enzyme-sensitive components in the liquid detergent from enzymes.

[0077] Examples of detergent components that are sensitive to enzymes, and that can be degraded by enzymes, include (relevant enzyme in parentheses): xanthan gum (xanthanase), steric bond polymers (lipase), hydrogenated castor oil (lipase) ), perfume (lipase), methyl ester sulfonate surfactants, cellulose and cellulose derivatives (eg, CMC) (cellulase), and dextrin and cyclodextrin (amylase).

[0078] Sensitive detergent ingredients can also be encapsulated and therefore stabilized in the microcapsules of the invention. Sensitive detergent ingredients are prone to degradation during storage. Such detergent ingredients include bleaching compounds, bleaching activators, perfumes, polymers, adjuvant, surfactants, etc.

[0079] Generally, the microcapsules of the invention can be used to separate incompatible components / compounds in detergents.

[0080] The addition of microcapsules to detergents can be used to influence the visual appearance of the detergent product, such as an opacifying effect (small microcapsules) or a clearly visible particle effect (large microcapsules). The microcapsules can also be colored.

[0081] Microcapsules can be used to reduce enzyme dust levels during the handling and processing of enzyme products.

[0082] Unless otherwise indicated, all percentages are indicated as percentage by weight (% w / w) in the entire application.

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[0083] Microcapsule: The microcapsules are typically produced by forming water droplets in a continuum that is not miscible with water, that is, typically preparing a water-in-oil emulsion, and subsequently forming the membrane by interfacial polymerization via the addition of a crosslinking agent. After eventual cure, the capsule can be collected and rinsed further and formulated by methods known in the art. The capsule formulation is subsequently added to the detergent.

[0084] The payload, the main constituents of the membrane and the possible additional component to be encapsulated are in the aqueous phase. In the continuum there are components that stabilize the water droplets towards coalescence (emulsifiers, emulsion stabilizers, surfactants, etc.) and the cross-linking agent is also added via the continuum.

[0085] The emulsion can be prepared by any method known in the art, for example, by mechanical agitation, drip processes, membrane emulsion, microfluids, sonication, etc. In some cases, simple mixing of the phases will automatically result in an emulsion, often referred to as self-emulsion. The use of methods that result in a small size distribution is an advantage.

[0086] The crosslinking agent (agents) is typically added subsequently to the emulsion, or directly or more typically by preparing a solution of the crosslinking agent in a solvent that is soluble in the continuous phase. The emulsion and the cross-linking agent or the solution thereof can be mixed by conventional methods used in the art, for example, by simple mixing or by carefully controlling the flows of the emulsion and the cross-linking agent solution by an in-line mixer.

[0087] In some cases, healing of the capsules is needed to complete the formation of the membrane. Curing is often simple agitation of the capsules for some time to allow the interfacial polymerization reaction to end. In other cases, membrane formation can be stopped by adding a reaction quencher.

[0088] The capsules can be subsequently modified, for example, by reacting components on the membrane to hinder or reduce flocculation of the particles in the detergent as described in WO 99/01534.

[0089] The capsules produced can be isolated or concentrated by methods known in the art, for example, by filtration, centrifugation, distillation or decantation of the capsule dispersion.

[0090] The resulting capsules can be further formulated, for example, by the addition of surfactants to give the product the desired properties for storage, transport and subsequent handling and addition to the detergent. Other microcapsule formulation agents include rheology modifiers, biocides (e.g., Proxel), acid / base for pH adjustment (which will also be adjusted within the microcapsules), and water for water activity adjustment.

[0091] The capsule formation process may include the following steps:

- Preparation of the initial phase (s) aqueous (s) and oily (s),

- Formation of a water-in-oil emulsion,

- Formation of the membrane by interfacial polymerization,

- Optional subsequent modification,

- Optional isolation and / or formulation,

- Addition to detergent.

[0092] The process may be either a discontinuous process or a continuous or semi-continuous process.

[0093] A microcapsule according to the invention is a small aqueous sphere with a constant membrane around it. The material within the microcapsule is referred to as the core, internal phase or filler, while the membrane is sometimes called a coating, coating or wall. The microcapsules of the invention have diameters between 0.5 | _im and 2 millimeters. Preferably, the average diameter of the microcapsules is in the range of 1 | _im to 1000 | _im, more preferably in the range of 5 | _im to 500 | _im, even more preferably in the range of 10 | _im to 500 | _im, even more preferably in the range of 50 | _im to 500 | _im, and most preferably in the range of 50 | _im to 200 | _im. Alternatively, the diameter of the microcapsules is in the range of 0.5 | _im to 30 | _im; or in the range of 1 | _im to 25 | _im. The diameter of the microcapsule is measured in the oily phase after the polymerization is complete. The diameter of the capsule may change depending on the water activity of the surrounding chemical environment.

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[0094] Enzyme microencapsulation, as used in the present invention, can be performed by interfacial polymerization, where the two reagents of a polymerization reaction meet at an interface and react rapidly. The basis of this method is a reaction of a polyamine with an acid derivative, usually an acid halide, which acts as a crosslinking agent. The polyamine is preferably substantially water soluble (when in the free base form). Under the right conditions, thin flexible membranes form quickly at the interface. One way to carry out the polymerization is to use an aqueous solution of the enzyme and the polyamine, which are emulsified with a non-aqueous solvent (and an emulsifier), and a solution with the acid derivative is added. An alkaline agent may be present in the enzymatic solution to neutralize the acid formed during the reaction. Polymeric (polyamide) membranes form instantly at the interface of the emulsion droplets. The polymer membrane of the microcapsule is typically of a cationic nature and, therefore, binds / complexes with compounds of an anionic nature.

[0095] The diameter of the microcapsules is determined by the size of the droplets of the emulsion, which is controlled, for example, by the rate of agitation.

[0096] Emulsion: An emulsion is a temporary or permanent dispersion of a liquid phase in a second liquid phase. The second liquid is generally referred to as the continuous phase. Surfactants are commonly used to aid in the formation and stabilization of emulsions. Not all surfactants are equally capable of stabilizing an emulsion. It is necessary to select the type and amount of a surfactant for optimum emulsion utility especially with respect to the preparation and physical stability of the emulsion, and the stability during dilution and further treatment. Physical stability refers to maintaining an emulsion in a dispersion form. Processes such as coalescence, aggregation, adsorption to container walls, sedimentation and cream formation are forms of physical instability, and should be avoided. Examples of suitable surfactants are described in WO 97/24177, pages 19-21; and in WO 99/01534.

[0097] The emulsions can be further classified or as simple emulsions, where the dispersed liquid phase is a simple homogeneous liquid, or as a more complex emulsion, where the dispersed liquid phase is a heterogeneous combination of liquid or solid phases, such as a double emulsion or a multiple emulsion. For example, a double emulsion of water in oil or multiple emulsion can be formed where the aqueous phase itself also contains an emulsified oily phase; This type of emulsion can be specified as an oil-in-water-in-oil emulsion (Oil / Water / Oil). Alternatively, a water-in-oil emulsion can be formed where the aqueous phase contains a dispersed solid phase often referred to as a suspension-emulsion. Other more complex emulsions can be described. Due to the inherent difficulty in describing such systems, the term emulsion is used to describe both simple and more complex emulsions without necessarily limiting the shape of the emulsion or the type and number of phases present.

[0098] Polyamine: the rigidity / flexibility and permeability of the membrane is mainly influenced by the choice of polyamine. The polyamine according to the invention is a polyramine polyamine. Each branch, which preferably ends with a primary amino group, serves as a mooring point in the membrane network, thus giving the favorable properties of the invention. A polyramine polyamine according to the present invention is a polyamine having more than two branching points and more than two reactive amino groups (capable of reacting with the crosslinking agent, that is, primary and secondary amino groups). Polyramide polyamine is used as a starting material when the emulsion is prepared - it is not formed in situ from other starting materials. To obtain the attractive properties of the invention, the polyramified structure of the polyamine must be presented as a starting material.

[0099] There is a close relationship between the number of branching points and the number of primary amines, since the primary amines will always be positioned at the end of a branch: a linear amine can only contain two primary amines. For each branch point hypothetically introduced into such a linear diamine, one or more primary amines will be allowed to be introduced at the end of the introduced branch (s). In this context we understand the primary amino group as part of the branch, that is, the end point of the branch. For example, we consider both tris (2- aminoethyl) amine and 1,2,3-propanotriamine as branching point molecules. For the invention, the polyamine has at least four primary amines. Branching points can be introduced from an aliphatic hydrocarbon chain as in the examples listed above or from unsaturated carbon bonds, such as in, for example, 3,3'-diaminobenzidine, or from tertiary amino groups, such as in N , N, N ', N'-tetrakis- (2- aminoethyl) ethylenediamine.

[0100] In addition to the number of branching points, we have found that the compactness of the reactive amino groups is of great importance. A substance such as, for example, N, N, N ', N'-tetrakis- (12-

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aminododecyl) ethylenediamine would not be suitable. Nor would a peptide or protein, such as an enzyme, be suitable for membrane formation. Therefore, polypyramified polyamine is not a peptide or protein.

[0101] In one embodiment, the reactive amino groups constitute at least 15% of the molecular weight of the polyramified polyamine, such as more than 20%, or more than 25%. Preferably, the molecular weight of the poly-branched polyamine is at least 1 kDa; more preferably, the molecular weight of the polyramified polyamine is at least 1.3 kDa.

[0102] In a preferred embodiment, the poly-branched polyamine is a polyethyleneimine (PEI), and modifications thereof, with more than two branching points and more than two reactive amino groups; where the reactive amino groups constitute at least 15% of the molecular weight of the PEI, such as more than 20%, or more than 25%. Preferably, the molecular weight of the PEI is at least 1 kDa.

[0103] The combinations of different polyramified polyamines can be used for the preparation of the microcapsule according to the invention.

[0104] The advantageous properties (for example, enzyme storage stability, reduced enzyme leakage, reduced inlet flow of detergent ingredients) of the microcapsule of the invention can be improved by adding one or more small amines with a lower molecular weight. at 1 kDa. The small amine is preferably substantially water-soluble (when in the free base form) and can be a material such as ethylenediamine, hexamethylenediamine, hexadiamine, diethylene tetramine, ethyltetramine, diaminobenzene, piperazine, tetramethylenepentamine or, preferably, diethylenetriamine (DETA). Small amines can be added in an amount of up to 50%, preferably up to 40%, up to 30%, up to 20%, up to 10%, or up to 5%, by weight of the total small amine content and polyramide polyamine, when the microcapsule of the invention is prepared.

[0105] Crosslinking agent: The crosslinking agent as used in the present invention is a molecule with at least two groups / sites capable of reacting with amines to form covalent bonds.

[0106] The crosslinking agent is preferably oil soluble and may be in the form of an acid anhydride or acid halide, preferably an acid chloride. For example, it may be adipoyl chloride, sebacoyl chloride, dodecanedioic acid chloride, phthaloyl chloride, terephthaloyl chloride, isophthaloyl chloride, or trimesoyl chloride; but, preferably, the crosslinking agent is terephthaloyl chloride or trimesoyl chloride.

[0107] The invention further relates to a method for washing an article, a method comprising the steps of:

to. Exposing an article to a wash solution comprising the inventive polypeptide or having DNase activity or a detergent composition comprising the polypeptide;

b. Complete at least one wash cycle; Y

C. Rinse the item optionally,

Where the article is a textile.

[0108] The pH of the liquid solution is in the range of 1 to 11, such as in the range of 5.5 to 11, such as in the range of 7 to 9, in the range of 7 to 8 or in the range range from 7 to 8.5.

[0109] The wash solution may have a temperature in the range of 5 ° C to 95 ° C, or in the range of 10 ° C to 80 ° C, in the range of 10 ° C to 70 ° C, in the range of 10 ° C to 60 ° C, in the range of 10 ° C to 50 ° C, in the range of 15 ° C to 40 ° C or in the range of 20 ° C to 30 ° C. In one embodiment, the temperature of the wash solution is 30 ° C.

[0110] In one embodiment of the invention, the method of washing an article further comprises draining the washing solution or part of the washing solution after completing a washing cycle. The wash solution can then be reused in a subsequent wash cycle or in a subsequent rinse cycle. The article may be exposed to the wash solution for a first and optionally a second or third wash cycle. In one embodiment, the article is rinsed after being exposed to the wash solution. The article can be rinsed with water or with water comprising a conditioner.

[0111] The invention further relates to an article washed according to the inventive method.

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[0112] The invention further relates to a pharmaceutical composition comprising a polypeptide having DNase activity and a pharmaceutical adjunct ingredient, wherein the polypeptide having DNase activity is obtained from a fungal source. The composition can be used to release or remove a biofilm or prevent biofilm formation.

[0113] The antiparasitic compound may be one or more of a benzazole, such as albendazole, mebendazole and thiabendazole; an azol, such as metronidazole and tinidazole; a macrocycle, such as amphotericin B, rifampin and ivermectin; Pirantel pamoato; diethylcarbamazine; niclosamide; praziquantel; melarsopro; and eflornithine.

[0114] The antiviral compound may be one or more of a nucleoside analogue reverse transcriptase inhibitor, such as acyclovir, didanosine, stavudine, cidovudine, lamivudine, abacavir, emtricitabine and entecavir; an uncoated inhibitor such as amantadine, rimantadine and pleconaril; a protease inhibitor such as saquinavir, ritonavir, indinavir, nelfinavir and amprenavir; zanamivir; oseltamivir; and rifampin. The antibacterial compound may be one or more of an aminoglycoside such as gentamicin, kanamycin and streptomycin; a betalactam such as penicillin, ampicillin and imipenemo; a cephalosporin such as ceftazidime, a quinolone such as ciprofloxacin; a macrolide such as azithromycin, clarithromycin, dithromycin, erythromycin, roxithromycin and telithromycin; an oxazolidinone such as linezolid; an ansamycin such as rifamycin; a sulfonamide; a tetracycline such as doxycycline; a glycopeptide such as vancomycin; sulfisoxazole, trimethoprim, novobiocin, daptomycin and linezolid.

[0115] The antifungal compound may be one or more of an azol, such as miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, sertaconazole, sulconazole, thioconazole, fluconazole, itraconazole, isavuconazole, ravonaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravconaconazole, ravonaconazole, ravconazole, ravconazole, posconazole , voriconazole, terconazole and abafungin; a macrocycle, such as natamycin, rimocidine, filipin, nystatin, amphotericin B, candicin, hamycin; an allyl amine such as terbinafine, naphthifine and butenaphine; an echinocandin such as andidulafungin, caspofungin and micafungin; or others such as polygodial, cyclopyrox, tolnaphtate, benzoic acid, undecylenic acid, flucytosine and griseofulvin.

[0116] The invention also relates to a permanent medical device characterized by the fact that at least a portion of a surface contactable with the patient of said device is coated with the pharmaceutical composition.

[0117] The device may be a catheter such as a central venous catheter, intravascular catheter, urinary catheter, Hickman catheter, peritoneal dialysis catheter, endrotracheal catheter, or where the device is a mechanical heart valve, a cardiac pacemaker, a shunt arteriovenous, a sclera closure, a joint prosthesis, a tympanostomy tube, a tracheotomy tube, a voice prosthesis, a penile prosthesis, an artificial urinary sphincter, a synthetic pubovaginal sling, a surgical suture, a bone anchor, a bone screw, an intraocular lens, a contact lens, an intrauterine device, an aortofemoral graft, a vascular graft, a needle, a Luer-Lok connector, a needleless connector or a surgical instrument.

[0118] The pharmaceutical composition can be formulated as a liquid, lotion, cream, spray, gel or ointment.

[0119] The pharmaceutical composition may be for administration to an animal patient. The animal patient may be a mammalian patient. The mammalian patient can be a human.

[0120] The polypeptide having DNase activity can be obtained from Aspergillus, for example, from Aspergillus oryzae. In one embodiment of the invention, the polypeptide having DNase activity is the claimed polypeptide.

[0121] One aspect of the present invention relates to isolated polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 60%, for example, at least 65%, at minus 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

[0122] In one embodiment, the present invention relates to isolated polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 60%, for example, at least 65 %, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SeQ iD #: 9.

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[0123] In one embodiment, the present invention relates to isolated polypeptides having a sequence identity with SEQ ID No. 8 of at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 8.

[0124] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 60% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0125] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 60% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0126] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 65% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0127] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 65% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0128] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 70% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0129] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 70% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0130] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 75% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0131] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 75% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0132] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 80% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0133] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 80% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0134] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 85% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0135] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 85% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0136] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 90% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

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[0137] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 90% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0138] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 91% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0139] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 91% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0140] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 92% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0141] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 92% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0142] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 93% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0143] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 93% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0144] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 94% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0145] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 94% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0146] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 95% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0147] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 95% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0148] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 96% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0149] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 96% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0150] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 97% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0151] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of at least 97% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

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[0152] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 98% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0153] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 98% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0154] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 99% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0155] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 99% having DNase activity and the polypeptide being use to prevent, reduce or eliminate a biofilm of an article.

[0156] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 2 of 100% having DNase activity and the polypeptide is used to prevent , reduce or eliminate a biofilm of an article.

[0157] In one embodiment, the present invention relates to polypeptides having a sequence identity with the mature polypeptide of SEQ ID No. 9 of 100% having DNase activity and the polypeptide is used to prevent , reduce or eliminate a biofilm of an article.

[0158] In one embodiment, the polypeptide has been isolated. A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or is a fragment thereof that has DNase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID No. 2. In another aspect, the polypeptide comprises or consists of amino acids 1 to 206 of SEQ ID No. 2.

[0159] In one embodiment, the polypeptide has been isolated. A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID No. 9 or an allelic variant thereof; or is a fragment thereof that has DNase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID No. 9. In another aspect, the polypeptide comprises or consists of amino acids 1 to 204 of SEQ ID No. 9. of the present invention refers to a composition comprising or consisting essentially of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 and a polypeptide of the present invention consisting of the amino acid sequence of the SEQ ID No.: 9.

[0160] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under low astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID N. °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0161] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under medium-high astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID No.: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0162] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under medium astringency conditions with (i) the mature polypeptide coding sequence of sEq ID N. °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0163] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under medium-high astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID No.: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0164] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under high astringency conditions with (i) the coding sequence

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of the mature polypeptide of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0165] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under conditions of very high astringency with (i) the mature polypeptide coding sequence of SEQ ID N . °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0166] The polynucleotide of SEQ ID No. 1 or a sub-sequence thereof, as well as the polypeptide of SEQ ID No. 2 or a fragment thereof or the polypeptide of SEQ ID No. °: 9 or a fragment thereof, can be used to design nucleic acid probes to identify and clone DNA encoding polypeptides that have DNase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, to identify and isolate the corresponding gene therein. Such probes may be considerably shorter than the entire sequence, but they should be at least 15 in length, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at at least 800 nucleotides, or at least 900 nucleotides. Both DNA and RNA probes can be used. The probes are typically labeled to detect the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are included in the present invention.

[0167] A genomic DNA or cDNA library obtained from other strains of this type can be screened for DNA that hybridizes with the probes described above and encodes a DNase activity polypeptide. Genomic or other DNA of other strains of this type can be separated by polyacrylamide or agarose gel electrophoresis, or other separation techniques. The DNA of the libraries or the separated DNA can be transferred to nitrocellulose or other suitable carrier material and immobilized therein. To identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a sub-sequence thereof, the carrier material is used in a Southern blot.

[0168] For the purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) the cDNA sequence thereof; (iv) the complement throughout its length; or (v) a sub sequence thereof; under conditions of very low-low astringency, conditions of low-medium astringency, conditions of medium astringency, conditions of medium-high astringency, conditions of high astringency to conditions of very high astringency. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other means of detection known in the art.

[0169] In another embodiment, the present invention relates to a polypeptide having DNase activity encoded by a polynucleotide having a sequence identity with the mature polypeptide coding sequence of SEQ ID No. 1 or cDNA sequence thereof of at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at minus 99%, or 100%. In another embodiment, the polypeptide has been isolated.

[0170] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, elimination, and / or insertion in one or more (eg, several) positions . In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 is up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acid changes may be of little relevance, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small extensions in the amino or carboxyl terminal, such as an aminoterminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0171] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 9 comprising a substitution, elimination, and / or insertion in one or more (eg, several) positions . In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 9 is up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acid changes may be of little relevance, that is, conservative amino acid substitutions or insertions that are

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significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small extensions of the amino or carboxyl terminal, such as an aminoterminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0172] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine) , aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

[0173] Alternatively, amino acid changes are such that the physicochemical properties of the polypeptides are altered. For example, amino acid changes can improve the thermostability of the polypeptide, alter substrate specificity, change the optimum pH, and the like.

[0174] Essential amino acids in a polypeptide can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the prior art, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are evaluated for DNase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with site amino acid mutation. of putative contact. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a relative polypeptide.

[0175] Substitutions, deletions and / or insertions of single or multiple amino acids can be made and evaluated using known methods of mutagenesis, recombination, and / or redistribution, followed by a relevant screening procedure, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that may be used include error-prone PCR, phage display (eg, Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) , and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0176] The mutagenesis / redistribution methods can be combined with automated high-performance screening methods to detect activity of mutagenized cloned polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and sequenced quickly using standard methods of the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0177] The polypeptide may be a hybrid polypeptide where a region of a polypeptide is fused in the N-terminal or the C-terminal of a region of another polypeptide.

[0178] The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide wherein another polypeptide is fused at the N-terminal or the C-terminal of the polypeptide of the present invention. A fusion polypeptide is produced by fusion of a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include linking the coding sequences encoding the polypeptides so that they are in the frame and that the expression of the fusion polypeptide is under the control of the same promoter (the same promoters) and terminator Fusion polypeptides can also be constructed using intein technology where fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779 ).

[0179] A fusion polypeptide may further comprise a cleavage site between the two polypeptides. After secretion of the fusion protein, the site is cleaved and the two polypeptides are released. Examples of cleavage sites include, but are not limited to, the sites described in Martin et al., 2003, J. Ind. Microbiol. Biotechnol 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol 63: 34883493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al.,

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1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Polynucleotides

[0180] The present invention also relates to polynucleotides encoding a polypeptide, as described herein. In one embodiment, the polynucleotide encoding the polypeptide of the present invention has been isolated. In another embodiment, the polynucleotide encoding a polypeptide of the present invention comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 12.

[0181] The techniques used to isolate or clone a polynucleotide are known in the art and include isolation of genomic DNA or cDNA, or a combination thereof. Cloning of genomic DNA polynucleotides can be performed, for example, using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural characteristics. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), linkage activated transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. The polynucleotides can be cloned from an Aspergillus strain, or a related organism and, therefore, for example, can be an allelic or species variant of the polypeptide encoding the polynucleotide region.

[0182] Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to forms of the polypeptide that do not occur naturally. These polypeptides may differ in some designed manner from the polypeptide isolated from its native source, for example, variants that differ in specific activity, thermostability, optimum pH, or the like. Variants can be constructed based on the polynucleotide presented as the mature polypeptide coding sequence of SEQ ID No. 1 or the cDNA sequence thereof, for example, a sub-sequence thereof, and / or by the introduction of nucleotide substitutions that do not imply a change in the amino acid sequence of the polypeptide, but that correspond to the use of the codon of the host organism destined for the production of the enzyme, or by the introduction of nucleotide substitutions that can give rise to a sequence different amino acids. For a general description of nucleotide substitution, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107. Thus, in one embodiment, the polynucleotide comprises or consists of the polynucleotide sequence set forth in SEQ ID No. 12, where the codons have been modified by nucleotide substitutions to correspond to the use of the codon of the host organism intended for the production of the polypeptide of the present invention.

Nucleic Acid Constructs

[0183] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the sequences. of control.

[0184] The polynucleotide can be manipulated in a variety of ways to provide expression of the polypeptide. Manipulation of the polynucleotide before insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0185] The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for the expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that shows transcriptional activity in the cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0186] Examples of promoters suitable for directing the transcription of the nucleic acid constructs of the present invention in a bacterial host cell are promoters obtained from the alpha-amylase (amyQ) gene of Bacillus amyloliquefaciens, the alpha- gene Bacillus licheniformis amylase (amyL), the Bacillus licheniformis penicillinase (penP) gene, the Bacillus stearothermophilus maltogenic amylase (amyM) gene, the Bacillus subtilis levansucrase (sacB) gene, the Baylillus xylA x xyl genes subtilis, the crylIIA gene of Bacillus thuringiensis (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), lac operon of E. coli, trc promoter of E. coli (Egon et al., 1988, Gene 69: 301- 315), the Streptomyces coelicolor agarase (dagA) gene, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727- 3731), like the tac promoter (DeBoer et al.,

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1983, Proc. Natl Acad. Sci. USA 80: 21-25). Other promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of serial promoters are described in WO 99/43835.

[0187] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, neutral alpha-amylase of Aspergillus niger, alpha-amylase stable in acid of Aspergillus niger, glucoamliase (glaA) of Aspergillus niger or Aspergillus awamori, TAKA amylase of Aspergillus oryzae, alkaline protease of Aspergillus oryzae, triose phosphate isomerase of Aspergillus oryzae, trypsin-like protease (96) ), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, beta-glucosidase from Rhizomucor miehei Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucan sa II from Trichoderma reesei, endoglucanase III from Trichoderma reesei, endoglucanase V from Trichoderma reesei, xylanase I from Trichoderma reesei, xylanase II from Trichoderma reesei, xylanase III from Trichoderma reesei, beta-xylosidase from Trichoderma reesei reesei from reicho from Trichoderma reesei, as well as the NA2-tpi promoter (a modified promoter of a neutral alpha-amylase gene from Aspergillus where the untranslated leader has been replaced by an untranslated leader of a triose phosphate isomerase gene from Aspergillus; Non-limiting examples include modified promoters of a neutral alpha-amylase gene from Aspergillus niger where the untranslated leader has been replaced by an untranslated leader of a triose phosphate isomerase gene from Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated and hybrid promoters thereof. Other promoters are described in US Pat. No. 6,011,147.

[0188] In a yeast host, useful promoters of the genes for enolase (ENO-1) of

Saccharomyces cerevisiae, galactokinase (GAL1) from Saccharomyces cerevisiae, alcohol

dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP) from Saccharomyces cerevisiae, triosa phosphate isomerase (TPI) from Saccharomyces cerevisiae, metallothionein (CUP1) from Saccharomyces cerevisiae, and 3- phosphoglycerate kinase from Saccharomyces cerevisiae. Other promoters useful for yeast host cells are described in Romanos et al., 1992, Yeast 8: 423-488.

[0189] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3 'terminal of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in the present invention.

[0190] Preferred terminators for bacterial host cells are obtained from the genes for alkaline protease (aprH) of Bacillus clausii, alpha-amylase (amyL) from Bacillus licheniformis, and ribosomal RNA (rrnB) from Escherichia coli.

[0191] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranylate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, TAKA amylase or similar protease amylase Trypsin from Fusarium oxysporum, beta-glucosidase from Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, cellobioiohydrolase II from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichoderma reesei, Endoglucanase III from Trichodermasalasease from Trichodermaelasemasalase-3 I from Trichoderma reesei, xylanase II from Trichoderma reesei, xylanase III from Trichoderma reesei, beta-xylosidase from Trichoderma reesei, and translation elongation factor from Trichoderma reesei.

[0192] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are described in Romanos et al., 1992, supra.

[0193] The control sequence may also be a mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases gene expression.

[0194] Examples of suitable mRNA stabilizing regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 34653471).

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[0195] The control sequence can also be a leader, an untranslated region of an mRNA that is important for translation by the host cell. The leader is operatively linked to the 5 'terminal of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell can be used.

[0196] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0197] Suitable leaders for yeast host cells are obtained from the genes for enochase (ENO-1) from Saccharomyces cerevisiae, 3-phosphoglycerate kinase from Saccharomyces cerevisiae, alpha factor from Saccharomyces cerevisiae, and alcohol dehydrogenase / glyceraldehyde-3- phosphate dehydrogenase (ADH2 / GAP) from Saccharomyces cerevisiae.

[0198] The control sequence may also be a polyadenylation sequence, a sequence operatively linked to the 3 'terminal of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0199] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae proteases, and similar protease, of Fusarium oxysporum.

[0200] Polyadenylation sequences useful for yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0201] The control sequence may also be a coding region of the signal peptide that encodes a signal peptide linked to the N-terminal of a polypeptide and directs the polypeptide to the secretory pathway of the cell. The 5 'end of the polynucleotide coding sequence may intrinsically contain a signal peptide coding sequence naturally linked in the reading frame of the translation with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5 'end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence can simply replace the natural signal peptide coding sequence to improve polypeptide secretion. However, any coding sequence of the signal peptide that directs the expressed polypeptide to the secretory pathway of a host cell can be used.

[0202] The signal peptide coding sequences effective for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, alpha-amylase Bacillus stearothermophilus, neutral proteases of Bacillus stearothermophilus (nprT, nprS, nprM), and prsA of Bacillus subtilis. Other signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0203] The signal peptide coding sequences effective for the filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for neutral Aspergillus niger amylase, Aspergillus niger glucoamylase, TAKA Aspergillus oryzae amylase, Humicola insole cellulase, Humicola insole cellulase Endoglucanase V from Humicola insolens, Humicola lanuginosa lipase, and aspartic proteinase from Rhizomucor miehei.

[0204] Signal peptides useful for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described in Romanos et al., 1992, supra.

[0205] The control sequence may also be a coding sequence of the propeptide encoding a propeptide positioned at the N-terminal of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted into an active polypeptide by catalytic or autocatalytic cleavage of the propolypeptide propeptide. The coding sequence of the propeptide can be obtained from the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and alpha factor from Saccharomyces cerevisiae.

[0206] When both the signal peptide and the propeptide sequences are present, the propeptide sequence is located next to the N-terminal of a polypeptide and the signal peptide sequence is placed next to the N-terminal of the propeptide sequence.

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[0207] It may also be desirable to add regulatory sequences that regulate polypeptide expression with respect to host cell growth. Examples of regulatory sequences are those that cause gene expression to be activated or deactivated in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase promoter I, and Trichoderma reesei II. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence.

Expression vectors

[0208] The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences can be linked to produce a recombinant expression vector that can include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by insertion of the polynucleotide or a nucleic acid construct comprising the polynucleotide into a vector suitable for expression. When creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to the appropriate control sequences for expression. In one embodiment, the expression vector comprises the polynucleotide sequence set forth in SEQ ID NO: 12 operably linked to the appropriate control sequences for expression. In a further embodiment, the codons of the polynucleotide sequence have been modified by nucleotide substitutions to correspond to the use of the codon of the host organism intended for the production of the polypeptide of the present invention. The recombinant expression vector can be any vector (for example, a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause polynucleotide expression. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a closed linear or circular plasmid.

[0209] The vector may be an autonomous replication vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or a chromosome. artificial. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated with the chromosome (the chromosomes) where it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon can be used.

[0210] The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced, or similar cells. A selectable marker is a gene whose product provides resistance to viruses or biocides, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0211] Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin resistance, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosilaminoimidazol-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase phosphate) , hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. The amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the Streptomyces hygroscopicus bar gene are preferred for use in an Aspergillus cell. AdeA, adeB, amdS, hph, and pyrG genes are preferred for use in a Trichoderma cell.

[0212] The selectable marker can be a double selectable marker system as described in WO 2010/039889. In one aspect, the double selectable marker is a hph-tk double selectable marker system.

[0213] The vector preferably contains an element (s) that allows the integration of the vector into the genome of the host cell or autonomous replication of the vector into the cell independent of the genome.

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[0214] For integration into the genome of the host cell, the vector may depend on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct homologous recombination integration into the genome of the host cell at a precise location (locations) on the chromosome (the chromosomes). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to improve the probability of homologous recombination. The integrational elements can be any sequence that is homologous to the target sequence in the genome of the host cell. In addition, the integrational elements may be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0215] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of the replication can be any plasmid replicator that mediates the autonomous replication that works in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0216] Examples of bacterial replication origins are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUB110, pE194, pTA1060, and pAM01 that allow replication in Bacillus.

[0217] Examples of origins of replication for use in a yeast host cell are the origin of 2 micron replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0218] Examples of useful origins of replication in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). The isolation of the AMA1 gene and the construction of plasmids or vectors comprising the gene can be achieved according to the methods described in WO 00/24883.

[0219] More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase the production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including a selectable marker gene amplifiable with the polynucleotide where cells containing amplified copies of the gene selectable marker and, therefore, additional copies of the polynucleotide, can be selected for cell culture in the presence of the appropriate selectable agent.

[0220] The methods used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, for example, Sambrook et al., 1989, supra).

Host cells

[0221] The present invention also relates to recombinant host cells, which comprise a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The term "host cell" encompasses any progeny of a progenitor cell that is not identical to the progenitor cell due to mutations that occur during replication. The choice of a host cell will depend largely on the gene encoding the polypeptide and its source.

[0222] The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, for example, a prokaryotic or a eukaryotic.

[0223] The prokaryotic host cell can be any gram-positive or gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include,

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but not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0224] The bacterial host cell can be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus lautus lautus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0225] The bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus.

[0226] The bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0227] The introduction of DNA into a Bacillus cell can be effected by transformation of protoplasts (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209221), electroporation (see, for example, Shigekawa and Dower, 1988 Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell can be effected by transformation of protoplasts (see, for example, Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell can be carried out by transformation of protoplasts, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, for example , Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, for example, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289- 6294). The introduction of DNA into a Pseudomonas cell can be performed by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell can be done by natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), transformation of protoplasts (see, for example, Catt and Jollick , 1991, Microbes 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol Rev. 45: 409-436). However, any method known in the art for the introduction of DNA into a host cell can be used.

[0228] The host cell can also be a eukaryotic, such as a mammalian, insect, plant or fungal cell.

[0229] The host cell may be a fungal cell. "Fungus," as used herein, includes the edges Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawkswort et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, United Kingdom).

[0230] The fungal host cell can be a yeast cell. "Yeast", as used herein, includes ascoesporogenic yeast (Endomycetales), basidiosporogenous yeast, and yeast of imperfect fungi (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0231] The yeast host cell may be a cell of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as a Kluyveromyces lactis cell, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, diastaticus Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.

[0232] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined in Hawkswort et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is due to hyphal lengthening and carbon catabolism is necessarily aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by grafting a single-celled talus and carbon catabolism can be fermentative.

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[0233] The filamentous fungal host cell can be a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Neceliocaphthora Nephioximix, Pencel , Phanerochaete, Phlebia, Pyromyces, Pleurotus, Schizofilum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.

[0234] For example, the filamentous fungal host cell may be a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkanderapps. pannocinta, rivulosa Ceriporiopsis, subrufa Ceriporiopsis, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium sulphureum, Fusarium sulphureum, Fusarium sulphureum, Fusarium sulphureum m torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus trichtemaderii, Trichtemaetemaderii, Tracystistemaderii, Trichormaemaeus, Tracyais , Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0235] Fungal cells can be transformed by a process that involves the formation of protoplasts, the transformation of protoplasts, and the regeneration of the cell wall in a manner known per se. Suitable procedures for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio / Technology 6: 1419-1422. Suitable methods for the transformation of Fusarium species are described in Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, volume 194, p. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl Acad. Sci. USA 75: 1920

Production methods

[0236] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) culturing a cell, which in its wild-type form produces the polypeptide, under conditions conducive to the production of the polypeptide; and optionally, (b) polypeptide recovery. In one aspect, the cell is an Aspergillus cell. In another aspect, the cell is an Aspergillus oryzae cell.

[0237] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) the cultivation of a recombinant host cell of the present invention under conditions conducive to the production of the polypeptide; and optionally, (b) polypeptide recovery.

[0238] Host cells are grown in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, cells can be cultured by shaking flask culture, or small-scale or large-scale fermentation (including, continuous fermentations, in batches, in fed batches, or in solid state) in the laboratory or in industrial fermenters in a suitable medium and under conditions that allow the polypeptide to be expressed and isolated. The culture takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (for example, in the catalogs of the American Type Culture Collection). If the polypeptide is secreted in the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0239] The polypeptide can be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzymatic product, or the disappearance of an enzyme substrate. For example, an enzymatic assay can be used to determine the activity of the polypeptide.

[0240] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures that include, but are not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.

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[0241] The polypeptide can be purified by a variety of methods known in the art that include, but are not limited to, chromatography (eg, ion exchange, affinity, hydrophobic, chromato-focus, and size exclusion), electrophoretic procedures (e.g. preparative isoelectric approach), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

[0242] In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention that expresses the polypeptide is used as a source of the polypeptide.

[0243] In one embodiment, the invention further comprises producing the polypeptide by culturing the recombinant host cell further comprising a polynucleotide encoding a second polypeptide of interest; preferably an enzyme of interest; more preferably a secreted enzyme of interest; even more preferably a hydrolase, isomerase, ligase, lyase, oxidoreductase, or a transferase; and most preferably the secreted enzyme is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitin, cutinase , cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucan-transferase, glucoamylase, invertase, lacasa, lipase, mannosidase, mutanasa, oxidase, pectinolytic enzyme, peroxidase, phytase, polypheoloxidase, ribase protease enzyme xylanase

[0244] In one embodiment, the second polypeptide of interest is heterologous or homologous to the host cell.

[0245] In one embodiment, the recombinant host cell is a fungal host cell; preferably a filamentous fungal host cell; more preferably a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Pacilomorice, Pompomorotinus, Pompomorotinus, Pomorotinus, Pyromus, Pyromus, Pompus , Schizofilum, Talaromyces, Thermoascus, Thielavia, Tolipocladium, Trametes, or Trichoderma; most preferably a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Cerpispsis Ceripus Ceriporiopsis subvermispora, inops Chrysosporium, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum , Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fus arium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes trichoderma, Trichodermata trichorumummata, Trichoderma trichitema, Trichoderma trichormata, Trichoderma trichormata , or Trichoderma viride.

[0246] In one embodiment, the recombinant host cell is a bacterial host cell; preferably a prokaryotic host cell; more preferably a gram-positive host cell; even more preferably a host cell of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces; and most preferably a host cell of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licillifususususususususususususususususususus, Bacillus subtilis, and Bacillus thuringiensis.

[0247] In one embodiment, a method of producing the second polypeptide of interest comprises culturing the host cell under conditions conducive to the production of the second polypeptide of interest.

[0248] In one embodiment, the method further comprises recovering the second polypeptide of interest. Fermentation broth formulations or cell compositions

[0249] The present invention also relates to a fermentation broth formulation or a cellular composition comprising a polypeptide of the present invention. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including host cells containing the gene encoding the polypeptide of the present invention that are used to produce the polypeptide of interest ), cell debris, biomass, fermentation media and / or fermentation products. In some

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embodiments, the composition is a complete broth of dead cells containing organic acid (s), dead cells and / or cell debris, and culture medium.

[0250] The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, fermentation broths are produced when microbial cultures are grown until saturation, incubated under limited carbon conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion in the culture medium mobile. The fermentation broth may contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is not fractionated and comprises the culture medium consumed and the cell debris present after the removal of the microbial cells (eg, filamentous fungal cells), for example, by centrifugation. In some embodiments, the fermentation broth contains consumed cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0251] In one embodiment, the fermentation broth formulation and cellular compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and / or a salt thereof and a second component of organic acid comprising at least one organic acid of 6 or more carbons and / or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the aforementioned and the second organic acid component is benzoic acid. , cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the aforementioned.

[0252] In one aspect, the composition contains an organic acid (s), and optionally also contains dead cells and / or cell debris. In one embodiment, dead cells and / or cell debris are removed from a complete broth of dead cells to provide a composition that is free of these components.

[0253] Fermentation broth formulations or cellular compositions may further comprise a preservative and / or antimicrobial agent (eg, bacteriostatic), which includes, but is not limited to, sorbitol, sodium chloride, potassium sorbate, and others. known in the art.

[0254] The complete dead cell broth or the composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the whole dead cell broth or composition contains the culture medium consumed and the cell debris present after the microbial cells (e.g., filamentous fungal cells) have been cultured until saturation and incubated under conditions of limited carbon for allow protein synthesis. In some embodiments, the complete dead cell broth or the composition contains the consumed cell culture medium, extracellular enzymes, and dead filamentous fungal cells. In some embodiments, the microbial cells present in the whole dead cell broth or the composition can be permeabilized and / or lysed using methods known in the art.

[0255] A complete broth or a cellular composition as described herein is typically a liquid, but may contain insoluble components, such as dead cells, cell debris, culture media components, and / or insoluble enzyme (s) ( s). In some embodiments, insoluble components can be removed to provide a clarified liquid composition.

[0256] The full broth formulations and cellular compositions of the present invention can be produced by a method described in WO 90/15861 or Wo 2010/096673.

Signal peptide

[0257] The present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids -37 to -16 of SEQ ID NO: 2. The present invention also relates to a polynucleotide encoding a propeptide comprising or consisting of amino acids -15 to -1 of SEQ ID NO: 2. The present invention also relates to a polynucleotide encoding a signal peptide and a propeptide comprising or consisting of amino acids -37 to -1 of SEQ ID No.: 2.

[0258] The polynucleotides may further comprise a gene encoding a protein, which is operably linked to the signal peptide and / or propeptide. The protein is preferably foreign to the signal peptide and / or propeptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 66 of SEQ ID NO: 1. In another

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aspect, the polynucleotide encoding the propeptide are nucleotides 67 to 111 of SEQ ID No. 1. In another aspect, the polynucleotide encoding the signal peptide and the propeptide are nucleotides 1 to 111 of SEQ ID N. °: 1.

[0259] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.

[0260] The present invention also relates to methods of producing a protein, which comprise (a) the cultivation of a recombinant host cell comprising such a polynucleotide; and (b) protein recovery.

[0261] The protein can be native or heterologous to a host cell. The term "protein" is not intended to refer to a specific length of the encoded product herein and, therefore, encompasses peptides, oligopeptides, and polypeptides. The term "protein" also encompasses two or more polypeptides combined to form the encoded product. Proteins also include hybrid polypeptides and fused polypeptides.

[0262] Preferably, the protein is a hormone, an enzyme, a receptor or a portion thereof, an antibody or a portion thereof, or an indicator. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, for example, an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase , carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, enzyme but pectinolitase, ribase, oxidase, protease, enzyme, ribosolase transglutaminase, or xylanase.

[0263] The gene can be obtained from any prokaryotic, eukaryotic or other source.

Enzyme of the invention - Deoxyribonuclease (DNase)

[0264] A polypeptide that has DNase activity or a deoxyribonuclease (DNase) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA structure, thereby degrading the DNA. The two terms polypeptide having DNase and DNase activity are used interchangeably.

[0265] According to the present invention, DNase can be obtained from a fungus; in particular, a DNase that can be obtained from an Aspergillus is preferred; In particular, a DNase that can be obtained from Aspergillus oryzae is preferred. In one embodiment of the present invention, the polypeptide having deoxyribonuclease activity is not the S1 nuclease of Aspergillus oryzae.

[0266] The DNase used in the present invention includes the mature polypeptide of SEQ ID NO: 2, shown as amino acids 1 to 206 of SEQ ID NO: 2, which are obtained from Aspergillus oryzae.

[0267] The polypeptide having DNase activity can be obtained from Aspergillus, for example, from Aspergillus oryzae. In one embodiment of the invention, the polypeptide having DNase activity is the claimed polypeptide.

[0268] One aspect of the present invention relates to isolated polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 2 of at least 60%, for example, at least 65%, at minus 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

[0269] In one embodiment, the present invention relates to isolated polypeptides having a sequence identity with the mature polypeptide of SEQ ID NO: 9 of at least 60%, for example, at least 65 %, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SeQ iD #: 9.

[0270] In one embodiment, the present invention relates to isolated polypeptides having a sequence identity with SEQ ID No. 8 of at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have DNase activity. In one aspect, the polypeptides differ by up to 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 8.

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[0271] A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or is a fragment thereof that has DNase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID No. 2. In another aspect, the polypeptide comprises or consists of amino acids 1 to 206 of SEQ ID No. 2.

[0272] In one embodiment, the polypeptide has been isolated. A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID No. 9 or an allelic variant thereof; or is a fragment thereof that has DNase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID No. 9. In another aspect, the polypeptide comprises or consists of amino acids 1 to 204 of SEQ ID No. 9. of the present invention refers to a composition comprising or consisting essentially of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 and a polypeptide of the present invention consisting of the amino acid sequence of the SEQ ID No.: 9.

[0273] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under low astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID N. °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). In one embodiment, the polypeptide has been isolated.

[0274] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under low-medium astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID No.: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0275] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under medium astringency conditions with (i) the mature polypeptide coding sequence of sEq ID N. °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0276] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under medium-high astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID No.: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0277] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under high astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID N. °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0278] In another embodiment, the present invention relates to an isolated polypeptide having DNase activity encoded by a polynucleotide that hybridizes under very high astringency conditions with (i) the mature polypeptide coding sequence of SEQ ID N . °: 1, (ii) the cDNA sequence thereof, or (iii) the full length complement of (i) or (ii). In one embodiment, the polypeptide has been isolated.

[0279] The polynucleotide of SEQ ID No. 1 or a sub-sequence thereof, as well as the polypeptide of SEQ ID No. 2 or a fragment thereof or the polypeptide of SEQ ID No. °: 9 or a fragment thereof, can be used to design nucleic acid probes to identify and clone polypeptides encoding DNA with DNase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, to identify and isolate the corresponding gene therein. Such probes may be considerably shorter than the entire sequence, but they should be at least 15 in length, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at at least 800 nucleotides, or at least 900 nucleotides. Both DNA and RNA probes can be used. The probes are typically labeled to detect the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are included in the present invention.

[0280] A library of genomic DNA or cDNA obtained from such other strains can be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having DNase activity. Genomic DNA

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or other such strains can be separated by polyacrylamide or agarose gel electrophoresis, or other separation techniques. Library DNA or separated DNA can be transferred and immobilized in nitrocellulose or other suitable carrier material. To identify a clone or aDn that hybridizes with SEQ ID NO: 1 or a sub-sequence thereof, the carrier material is used in a Southern blot.

[0281] For the purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) the cDNA sequence thereof; (iv) the complement throughout its length; or (v) a sub sequence thereof; under conditions of very low, low astringency, conditions of medium-low astringency, conditions of medium astringency, conditions of medium-high astringency, conditions of high astringency to conditions of very high astringency. The molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, x-ray film or any of the other detection means known in the art.

[0282] In another embodiment, the present invention relates to a polypeptide having DNase activity encoded by a polynucleotide with a sequence identity with the mature polypeptide coding sequence of SEQ ID No. 1 or the sequence cDNA thereof of at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In another embodiment, the polypeptide has been isolated.

[0283] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, a deletion, and / or an insertion into one or more (for example, several ) positions. In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 is up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acid changes may be of little relevance, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small extensions in the amino or carboxyl terminal, such as a methionine residue in the amino terminal; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0284] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 9 which comprise a substitution, elimination, and / or insertion in one or more (eg, several) positions . In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 9 is up to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Amino acid changes may be of little relevance, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small extensions in the amino and carboxyl terminal, such as a methionine residue in the amino terminal; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0285] The DNase enzyme may comprise or consist of the amino acid sequence shown as amino acids -37 to 206 of SEQ ID NO: 2 or a fragment thereof having DNase activity, such as the mature polypeptide . Or the DNase enzyme may comprise or consist of a fragment of amino acids -37 to 206 of SEQ ID No. 2 or amino acids 1 to 206 of SEQ ID No. 2, fragment for which one or more Amino acids are removed from the amino and / or carboxyl terminal of SEQ ID NO: 2.

[0286] The DNase enzyme may comprise or consist of the amino acid sequence shown as amino acids 1 to 204 of sEq ID No. 9 or a fragment thereof having DNase activity, such as the mature polypeptide. Or the DNase enzyme may comprise or consist of a fragment of amino acids 1 to 204 of SEQ ID No. 9 or amino acids 1 to 204 of SEQ ID No. 9, fragment for which one or more Amino acids are removed from the amino and / or carboxyl terminal of SEQ ID No. 9.

[0287] The present invention also provides DNase polypeptides that are substantially homologous to the above polypeptides, and homologous species (paralogs or orthologs) thereof. The term "substantially homologous" is used herein to denote polypeptides that are at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identical, and most preferably at least 99% or more identical to the amino acid sequence of SEQ ID No. 2 or the amino acid sequence of SEQ ID No. 9, or a fragment thereof that has DNase activity, or its orthologs or paralogs.

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[0288] In another embodiment, the DNase of SEQ ID NO: 2 comprises a substitution, elimination, and / or insertion in one or more (eg, several) positions. In another embodiment, the DNase of SEQ ID NO: 9 comprises a substitution, elimination, and / or insertion in one or more (eg, several) positions. In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID No. 2 or in the mature polypeptide of SEQ ID No. 9 is not greater than 10, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9. Amino acid changes may be of little relevance, that is, they are conservative amino acid substitutions or insertions that do not significantly affect folding and / or protein activity; small deletions, typically 1-30 amino acids; small extensions in the amino or carboxyl terminal, such as a methionine residue in the amino terminal; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or other function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0289] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine) , aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter the specific activity are known in the art and are described, for example, in H. Neurath and R.L. Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg , Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0290] Alternatively, amino acid changes are such that the physicochemical properties of the polypeptides are altered. For example, amino acid changes can improve the thermostability of the polypeptide, alter substrate specificity, change the optimum pH, and the like.

[0291] Essential amino acids in a polypeptide can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the prior art, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are evaluated for DNase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 46994708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as magnetic resonance imaging. nuclear, crystallography, electron diffraction, or photoaffinity marking, together with amino acid mutation of the putative contact site. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS. Lett 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a relative polypeptide.

[0292] Substitutions, deletions and / or insertions of single or multiple amino acids can be made and evaluated using known methods of mutagenesis, recombination, and / or redistribution, followed by a relevant screening procedure, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that may be used include error-prone PCR, phage display (eg, Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) , and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0293] The mutagenesis / redistribution methods can be combined with automated high-performance screening methods to detect activity of mutagenized cloned polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and sequenced quickly using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0294] The polypeptide may be a hybrid polypeptide in which a region of a polypeptide is fused in the N-terminal or the C-terminal of a region of another polypeptide.

[0295] The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide wherein another polypeptide is fused at the N-terminal or the C-terminal of the polypeptide of the present invention. A fusion polypeptide is produced by fusion of a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include linking the coding sequences encoding the polypeptides so that they are in the frame and that the expression of the fusion polypeptide is under the control of the same promoter (the same promoters) and terminator Fusion polypeptides can also be constructed using

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intein technology where fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0296] A fusion polypeptide may further comprise a cleavage site between the two polypeptides. In the secretion of the fusion protein, the site divides and releases the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites described in Martin et al., 2003, J. Ind. Microbiol. Biotechnology 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0297] The concentration of DNase in the wash solution is typically in the range of 0.00004-100 ppm of enzymatic protein, such as in the range of 0.00008-100, in the range of 0.0001-100 , in the range of 0.0002-100, in the range of 0.0004-100, in the range of 0.0008-100, in the range of 0.001-100 ppm of enzymatic protein, 0.01100 ppm of enzymatic protein , preferably 0.05-50 ppm enzymatic protein, more preferably 0.150 ppm enzyme protein, more preferably 0.1-30 ppm enzyme protein, more preferably 0.520 ppm enzyme protein, and most preferably 0.5- 10 ppm enzymatic protein.

[0298] The DNase of the present invention can be added to a detergent composition in an amount corresponding to at least 0.002 mg of DNase protein, such as at least 0.004 mg of DNase protein, at least 0.006 mg of DNase protein , at least 0.008 mg of DNase protein, at least 0.01 mg of DNase protein, at least 0.1 mg of protein, preferably at least 1 mg of protein, more preferably at least 10 mg of protein, even more preferably at least 15 mg of protein, most preferably at least 20 mg of protein, and even more preferably at least 25 mg of protein. Thus, the detergent composition may comprise at least 0.00008% of DNase protein, preferably at least 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.008%, 0.01%, 0.02% , 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% DNase protein.

[0299] The DNase of the detergent composition of the invention can be stabilized using conventional stabilizing agents, for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative , for example, an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenylboronic acid, and the composition can be formulated as described in, for example, WO92 / 19709 and WO92 / 19708.

[0300] A polypeptide of the present invention can also be incorporated into the detergent formulations described in WO97 / 07202, which is incorporated herein by reference.

Detergent compositions

[0301] In one embodiment, the invention is directed to detergent compositions that include an enzyme of the present invention in combination with one or more additional cleaning composition components. In one embodiment, the detergent composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ iD #: 9. It is also described that the detergent comprises a polypeptide consisting of the amino acid sequence of SEQ ID N .: 8 and a polypeptide of the present invention consisting of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the detergent composition is in solid form. In another embodiment, the detergent composition is in a liquid form. The choice of additional components is within the skill of the expert and includes conventional ingredients, including exemplary non-limiting components set forth below.

Liquid detergent composition

[0302] The liquid detergent composition may comprise a microcapsule of the invention, and thus form part of any detergent composition in any form, such as liquid and powder detergents, and soap and detergent tablets.

[0303] In one embodiment, the invention is directed to liquid detergent compositions comprising a microcapsule, as described above, in combination with one or more additional cleaning composition components.

[0304] The microcapsule, as described above, can be added to the liquid detergent composition in an amount corresponding to 0.0001% to 5% (w / w) active enzyme protein (AEP); preferably from

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0.001% to 5%, more preferably 0.005% to 5%, more preferably 0.005% to 4%, more preferably 0.005% to 3%, more preferably 0.005% to 2%, even more preferably 0.01% at 2%, and most preferably from 0.01% to 1% (w / w) active enzyme protein.

[0305] The liquid detergent composition has a physical form, which is not solid (or gaseous). It can be a pouring liquid, a paste, a pouring gel or a non-pouring gel. It can be either isotropic or structured, preferably isotropic. It can be a useful formulation for washing in automatic washing machines or for hand washing. It can also be a personal care product, such as a shampoo, toothpaste, or hand soap.

[0306] The liquid detergent composition may be aqueous, and typically contains at least 20% by weight and up to 95% water, such as up to 70% water, up to 50% water, up to 40% water. water, up to 30% water, or up to 20% water. Other types of liquids, which include, but are not limited to, alkanols, amines, diols, ethers and polyols, may be included in an aqueous liquid detergent. An aqueous liquid detergent may contain 0-30% organic solvent. A liquid detergent may even be non-aqueous, where the water content is less than 10%, preferably less than 5%.

[0307] The detergent ingredients can be physically separated from each other by compartments in water-soluble bags. Thus, the negative storage interaction between the components can be avoided. Different dissolution profiles of each of the compartments can also result in delayed dissolution of the selected components in the wash solution.

[0308] The detergent composition may take the form of a unit dose product. A unit dose product is the packaging of an individual dose in a non-reusable container. Its use in laundry detergents is on the rise. A unit dose product of detergent is the packaging (for example, in a bag made of a water-soluble film) of the amount of detergent used for a single wash.

[0309] The bags may be of any shape, shape and material that is suitable for the retention of the composition, for example, without allowing the release of the composition of the bag before contact with water. The bag is made of water-soluble film that includes an internal volume. Said internal volume can be divided into compartments of the bag. Preferred films are polymeric materials, preferably polymers that are formed in a film or sheet. Preferred polymers, copolymers or derivatives thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxymethyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, malt dextrin, polymethyl acryloxy ethyl cellulose polymethyl acrylate (HPMC). Preferably the level of polymer in the film, for example, PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The films may also be mixing compositions comprising mixtures of hydrolytically degradable and water-soluble polymers such as polyamide and polyvinyl alcohol (known under trade reference M8630 sold by Chris Craft In. Prod. Of Gary, Ind., USA) plus plasticizers such as glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The bags may comprise a cleaning composition for solid laundry or partial components and / or a liquid cleaning composition or partial components separated by the water-soluble film. The compartment for liquid components may have a different composition from that of the compartments containing solids (see, for example, US 2009/0011970).

[0310] The choice of detergent components may include, for textile care, consideration of the type of textile to be cleaned, the type and / or degree of dirt, the temperature at which the cleaning is to be carried out, and the formulation of the detergent product. Although the components mentioned below are categorized by general heading according to a particular functionality, this should not be construed as a limitation, since a component may comprise additional functionalities as will be appreciated by the person skilled in the art.

[0311] The choice of additional components is within the skill of the expert and includes conventional ingredients, including exemplary non-limiting components set forth below.

Surfactants

[0312] The detergent composition may comprise one or more surfactants, which may be anionic and / or cationic and / or non-ionic and / or semipolar and / or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant (the surfactants) are typically present at a level of from about 0.1% to 60% by weight, such as from about 1% to about 40%, or from about 3% to about 20%, or from about 3% to about 10%. The surfactant (the surfactants)

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It is chosen based on the desired cleaning application, and may include any conventional surfactant (any surfactants) known in the art.

[0313] When included therein, the detergent will normally contain from about 1% to about 40% by weight of an anionic surfactant, such as from about 5% to about 30%, with about 5% at about 15%, or from about 15% to about 20%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulphonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefinsulfonates, alkenosulfonates, 2,3-alkanesulphonates, alkano-sulfonates, alkano-sulfonates, 2,3-alkanesulfonates, alkano-sulphonates, 2,3-alkanesulfonates (sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, known also as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulphonates (PS), ester sulphonates, sulphonated fatty acid glycerol esters, alpha-sulphonated fatty acid methyl esters (alpha- SFMe or SES) including methyl ester sulfonate (MES), alkyl or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), derivatives of fatty acid amino acids, diesters and monoesters of sulfosuccinic acid or salt of fatty acids (soap), and combinations thereof.

[0314] When included therein, the detergent will normally contain from about 1% to about 40% by weight of a cationic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12% or from about 10% to approximately 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine cuat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldystearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, quaternary quaternary ammonium quaternary ammonium compounds and combinations thereof.

[0315] When included therein, the detergent will normally contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols, alkyl esters of alkoxylated fatty acids (PFA), such as alkyl esters of ethoxylated and / or propoxylated fatty acids, ethoxylates of alkylphenol, nonylphenol ethoxylates (NPE), alkyl polyglucosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (propoxylated fatty acid monoethanolamides) , polyhydroxyalkyl fatty acid amides, or N-acyl derivatives N-alkyl glucosamine (glucamides, GA, or fatty acid glucamides, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations of the same.

[0316] When included therein, the detergent will normally contain from about 0% to about 40% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxide, N- (coconut alkyl) -N, N-dimethylamine and N- (tallow alkyl) -N, N-bis (2 -hydroxyethyl) amine, and combinations thereof.

[0317] When included therein, the detergent will normally contain from about 0% to about 40% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethyl betaines, sulfobetaines, and combinations thereof.

Hydrotropes

[0318] A hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or conversely, polar substances in a non-polar environment). Typically, hydrotropes have both a hydrophilic and hydrophobic character (referred to as amphiphilic properties as surfactants are known); however, the molecular structure of hydrotropes generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not show a critical concentration above which self-aggregation occurs as observed for surfactants and lipids that form micellar, laminar or other well-defined mesophases. Instead, many hydrotropes

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they show a continuous type aggregation process where aggregate sizes grow as the concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing polar and non-polar substances, which include mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used in industries from pharmaceutical, personal care, food, to technical applications. The use of hydrotropes in detergent compositions allows, for example, more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesirable phenomena such as phase separation or high viscosity.

[0319] The detergent may contain 0-10% by weight, for example, 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents can be used. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthale, hydroxynaphthale of sodium, sodium ethylhexyl sulfate, and combinations thereof.

Adjuvants and adjuvants

[0320] The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of an adjuvant or detergent adjuvant or a mixture thereof. The adjuvant and / or the adjuvant can be particularly a chelating agent that forms water-soluble complexes with Ca and Mg. Any adjuvant and / or adjuvant known in the art for use in detergents can be used. Non-limiting examples of adjuvants include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, stratified silicates (eg, SKS-6 from Hoechst), ethanolamines such as 2-aminoethane-1-ol (MEA), diethanolamine (DEA, also known as 2,2'-iminodietan-1-ol), triethanolamine (TEA, also known as 2,2 ', 2 "-nitrilotrietan-1-ol), and (carboxymethyl) inulin (CMI), and combinations thereof.

[0321] The detergent composition may also contain from 0-50% by weight, such as about 5% to about 30%, of a detergent builder. The detergent composition may include an adjuvant alone, or in combination with an adjuvant, for example, a zeolite adjuvant. Non-limiting examples of adjuvants include polyacrylate homopolymers or copolymers thereof, such as poly (acrylic acid) (PAA) or acrylic / maleic acid (PAA / PMA) copolymer. Other non-limiting examples include citrate, chelants such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl or alkenyl succinic acid. Additional specific examples include 2,2 ', 2 "-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-W, W'-disuccinic acid (EDDS), acid methylglycinadiacetic acid (MGDA), W-glutamic acid, W-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra (methylenephosphonic acid) (EDTMPA), diethylenetriaminopentachis (methylene phosphonic acid) (DPAPA or DMPA) ), W- (2-hydroxyethyl) iminodiacetic acid (EDG), aspartic acid-W-monoacetic acid (ASMA), aspartic acid-W, W-diacetic acid (ASDA), aspartic acid-W-monopropionic acid (ASMP) , iminodisuccinic acid (IDA), W- (2-sulfomethyl) -aspartic acid (SMAS), W- (2-sulfoethyl) -aspartic acid (SEAS), W- (2-sulfomethyl) -glutamic acid (SMGL), acid W- (2-sulfoethyl) -glutamic (SEGL), W-methyliminodiacetic acid (MIDA), a-alanine-W, W-diacetic acid (a-ALDA), serine acid- W, W-diacetic (SEDA), isoserine-W, W-diacetic acid (ISDA), phenylalanine-W, W-diacetic (PHDA), anthranilic-W, W-diacetic (ANDA), sulphanilic acid-W, W-diacetic (SLDA), taurine-W, W-diacetic (tuda) and sulfomethyl-W, W-diacetic (SmDa), W- (2-hydroxyethyl) ethylenediamine-W, W ', W "-triacetic acid (HEDTA), dietanol glycine (DEG), diethylenetriamine penta (methylene phosphonic acid) (DTPMP), aminotris (methylene phosphonic acid) (ATMP), and combinations and salts thereof. Additional exemplary adjuvants and / or adjuvants are described in, for example, WO 09/102854, US 5977053

Bleaching systems

[0322] The detergent may contain 0-30% by weight, such as about 1% to about 20%, of a bleaching system. Any bleaching system known in the art for use in detergents can be used. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, sources of hydrogen peroxide such as sodium percarbonate, sodium perborates and hydrogen urea peroxide (1: 1), preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, diperoxy dicarboxylic acids, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono or tetrahydrate), salts of percarbonate, persulfate, perfosphate,

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persilicate, in combination with a bleach activator that forms peracids. The term "bleach activator" is understood herein as a compound that reacts with hydrogen peroxide to form a peracid via perhydrolysis. The peracid thus formed constitutes the activated bleach. Bleach activators suitable for use herein include those of the class of esters, amides, imides or anhydrides. Suitable examples are tetraacetylethylenediamine, (TAED) 4 - [(3,5,5-trimethylhexanoyl) oxy] benzene-1-sulphonate (ISONOBS), 4- (dodecanoyloxy) benzene-1-sulfonate (LOBS), 4- ( Decanoyloxy) benzene-1-sulfonate, 4- (decanoyloxy) benzoate (DOBS or DOBA), 4- (nonanoyloxy) benzene-1-sulphonate (NOBS), and / or those described in WO98 / 17767. A particular family of bleach activators of interest was described in EP624154 and triethyl acetyl citrate (ATC) is particularly preferred in that family. ATC or a short chain triglyceride such as triacetin has the advantage of being environmentally friendly. In addition, triethyl acetylcitrate and triacetin have good hydrolytic stability in the product after storage and are effective bleaching activators. Finally, ATC is multifunctional, since the citrate released in the perhydrolysis reaction can function as an adjuvant. Alternatively, the bleaching system may comprise peroxyacids of the type, for example, amide, imide, or sulfone. The bleaching system may also comprise peracids such as 6- (phthalimido) peroxyhexanoic acid (PAP). The bleaching system may also include a bleaching catalyst. In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulas:

image 1

where each R1 is independently a branched alkyl group containing from 9 to 24 carbons or a linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or a linear alkyl group containing 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl 2- hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl . Other exemplary bleaching systems are described, for example, in WO2007 / 087258, WO2007 / 087244,

WO2007 / 087259, EP1867708 (Vitamin K) and WO2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc or aluminum phthalocyanines.

[0323] Preferably, the bleach component comprises a source of peracid in addition to the bleach catalyst, particularly an organic bleach catalyst. The source of peracid can be selected from (a) preformed peracid; (b) percarbonate, perborate or persulfate salt (source of hydrogen peroxide) preferably in combination with a bleach activator; and (c) enzyme perhydrolase and an ester to form peracid in situ in the presence of water in a textile treatment step.

Polymers

[0324] The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents can be used. The polymer can function as an adjuvant as mentioned above, or it can provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, grease cleaning and / or defoamer properties. Some polymers may have more than one of the aforementioned properties and / or more than one of the reasons mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), poly (ethylene glycol) or poly (ethylene oxide) (PEG), ethoxylated poly (ethyleneimine), carboxymethylinulin (CMI), and polycarboxylates such as PAA, PAA / PMA polyaspartic acid, and copolymers of lauryl methacrylate / acrylic acid, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly (terephthalate of ethylene) and poly (oxyethylene terephthalate) (PET-POET), PVP, poly (vinylimidazole) (PVI), poly (vinylpyridine-W-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Additional exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are described in, for example, WO 2006/130575. The salts of the aforementioned polymers are also contemplated.

Fabric Matting Agents

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[0325] The detergent compositions of the present invention may also include tissue matting agents such as dyes or pigments, which, when formulated in detergent compositions, can be deposited on a tissue when said tissue is contacted with a solution of washing comprising said detergent compositions and thus altering the dye of said tissue by absorbing / reflecting visible light. Fluorescent bleaching agents emit at least some visible light. In contrast, tissue matting agents alter the dye of a surface since they absorb at least a portion of the visible light spectrum. Suitable tissue matting agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes included in the Color Index (CI) in the classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Blue Basic, Basic Violet and Basic Red, or mixtures thereof, for example, as described in WO2005 / 03274, WO2005 / 03275, WO2005 / 03276 and EP1876226 (which are incorporated herein by reference). The detergent composition preferably comprises from about 0.00003% by weight to about 0.2% by weight, from about 0.00008% by weight to about 0.05% by weight, or even from about 0, 0001% by weight to about 0.04% by weight of tissue matting agent. The composition may comprise from 0.0001% by weight to 0.2% by weight of tissue matting agent, this may be especially preferred when the composition is in the form of a unit dose bag. Suitable shading agents are also described in, for example, WO 2007/087257 and WO2007 / 087243.

Enzymes

[0326] The detergent additive as well as the detergent composition may comprise one or more additional enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, for example, a laccase, and / or peroxidase.

[0327] In general, the properties of the selected enzyme (s) should be compatible with the selected detergent, (ie, optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and The enzyme (s) should be present in effective amounts. In a preferred embodiment, the detergent additive or detergent composition comprises a protease. In another embodiment, said protease has at least 90%, such as at least 95%, such as 100% sequence identity with SEQ ID NO: 10

Cellulases

[0328] Suitable cellulases include those of bacterial or fungal origin. Chemically modified or designed protein mutants are included. Suitable cellulases include cellulases of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum described in US 4,435,307, US 5,648,278, US 5,648,278, US 5,648,278, US 5,648,278, US 5,648,278, US 5,648,278, US 5,648,278, WO 89/09259.

[0329] Especially suitable cellulases are alkaline or neutral cellulases that have color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and WO99 / 001544.

[0330] Other cellulases are the endo-beta-1,4-glucanase enzyme that has a sequence with at least 97% identity with the amino acid sequence from position 1 to position 773 of SEQ ID No.: 2 of WO 2002/099091 or a xyloglucanase of family 44, xyloglucanase enzyme having a sequence with at least 60% identity with positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.

[0331] Commercially available cellulases include Celluzyme ™, and Carezyme ™ (Novozymes A / S) Carezyme Premium ™ (Novozymes A / S), Celluclean ™ (Novozymes A / S), Celluclean Classic ™ (Novozymes A / S), Cellusoft ™ (Novozymes A / S), Whitezyme ™ (Novozymes A / S), Clazinase ™, and Puradax HA ™ (Genencor International Inc.), and KAC-500 (B) ™ (Kao Corporation).

Proteases

[0332] Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, for example, plant or microbial origin. Microbial origin is preferred. Chemically modified or designed protein mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease can, for example, be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A

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Metalloprotease protease may, for example, be a thermolysin of, for example, the M4 family or other metalloprotease such as those of the m5, M7 or M8 families.

[0333] The term "subtylases" refers to a subgroup of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. Subtylases can be divided into 6 subdivisions, that is, the subtilisin family, the termitase family, the K proteinase family, the lantibiotic peptidase family, the kexin family and the pyrolysin family.

[0334] Examples of subtylases are those obtained from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US7262042 and WO09 / 021867, and subtilisina lentus, subtilisina Notilisina, subtilisina Notilisina, subtilisina Notilisina , Bacillus licheniformis, subtilisin BPN ', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89 / 06279 and PD138 protease described in (WO93 / 18140). Other useful proteases may be those described in WO92 / 175177, WO01 / 016285, WO02 / 026024 and WO02 / 016547. Examples of trypsin-like proteases are trypsin (for example, of porcine or bovine origin) and the Fusarium protease described in WO89 / 06270, WO94 / 25583 and wO05 / 040372, and the chymotrypsin proteases obtained from Cellumonas described in WO05 / 052161 and WO05 / 052146.

[0335] An additional preferred protease is the Bacillus lentus DSM 5483 alkaline protease, as described, for example, in WO95 / 23221, and variants thereof, which are described in WO92 / 21760, WO95 / 23221, EP1921147 and EP1921148.

[0336] Examples of metalloproteases are neutral metalloprotease as described in WO07 / 044993 (Genencor Int.) Such as those obtained from Bacillus amyloliquefaciens.

[0337] Examples of useful proteases are the variants described in: WO92 / 19729, WO96 / 034946, WO98 / 20115, WO98 / 20116, WO99 / 011768, WO01 / 44452, WO03 / 006602, WO04 / 03186, WO04 / 041979, WO07 / 006305, WO11 / 036263, WO11 / 036264, especially variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97 , 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222 , 224, 232, 235, 236, 245, 248, 252 and 274 using the BPN 'numbering. More preferred, subtylase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, * 36D, V68A, N76D, N87S, R, * 97E, A98S, S99G, D, A, S99AD, S101G, M, R S103A, V104I, Y, N, S106A, G118V, R, H120D, N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D22, N218D22, N218D22, N218D22, N218D, N218D22 , Q236H, Q245R, N252K, T274A (using BPN 'numbering).

[0338] Suitable commercially available protease enzymes include those sold under the trade names of Alcalase®, DuralaseTm, DurazymTm, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase® , Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A / S), those sold under the trade name of Maxatase®, Maxacal®, Maxapem®, Purafect®, Prime ® Purafect, PreferenzTm, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, EffectenzTm, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco / DuPont), Axapem ™ (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US5352604) and variants thereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and cutinases

[0339] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein designed mutant enzymes are included. Examples include Thermomyces lipase, for example, from T. lanuginosus (previously called Humicola lanuginosa) as described in EP258068 and EP305216, Humicola cutinase, for example, H. insolens (WO96 / 13580), lipase from strains of Pseudomonas (some of these now renamed Burkholderia), for example, P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), strain of P. sp. SD705 (WO95 / 06720 and WO96 / 27002), P. wisconsinensis (WO96 / 12012), Streptomyces lipases of the GDSL type (WO10 / 065455), Magnaporthe grisea cutinase (WO1 0/1 07560), Pseudomonas mendocina cutinase (US5 , 389,536), Thermobifida fusca lipase (WO11 / 084412), Geobacillus stearothermophilus lipase (WO11 / 084417), Bacillus subtilis lipase (WO11 / 084599), and Streptomyces griseus lipase (WO11 / 150157) and S. pristinaes (WO. / 137147).

[0340] Other examples are lipase variants such as those described in EP407225, WO92 / 05249, WO94 / 01541, WO94 / 25578, WO95 / 14783, WO95 / 30744, WO95 / 35381, WO95 / 22615, WO96 / 00292, WO97 / 04079, WO97 / 07202, WO00 / 34450, WO00 / 60063, WO01 / 92502, WO07 / 87508 and WO09 / 109500.

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[0341] Preferred commercial lipase products include Lipolase ™, Lipex ™; Lipolex ™ and Lipoclean ™ (Novozymes A / S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

[0342] Other examples are lipases sometimes referred to as acyltransferases or perhydrolases, for example, acyltransferases homologous with the Candida lipase A of anthractic (WO10 / 111143), acyltransferase of Mycobacterium smegmatis (WO05 / 56782), perhydrolase family 7 (WO09 / 67279), and variants of M. smegmatis perhydrolase in particular the S54V variant used in the commercial Gentle Power Bleach product of Huntsman Textile Effects Pte Ltd (WO10 / 100028).

Amylases

[0343] Suitable amylases that can be used together with the enzyme of the invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or designed protein mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, for example, a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839.

[0344] Suitable amylases include amylases that have SEQ ID No. 2 in WO 95/10603 or variants that have 90% sequence identity with SEQ ID No. 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID No. 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: , 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201,202, 207, 208, 209, 211,243, 264, 304, 305, 391,408, and 444.

[0345] Different suitable amylases include amylases having SEQ ID No. 6 in WO 02/010355 or variants thereof that have 90% sequence identity with SEQ ID No. 6. Preferred variants of SEQ ID No.: 6 are those that have a deletion at positions 181 and 182 and a substitution at position 193.

[0346] Other suitable amylases are the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID No. 6 of WO 2006/066594 and residues 36 -483 of the B. licheniformis alpha-amylase shown in SEQ ID No. 4 of WO 2006/066594 or variants that have 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those that have a substitution, a deletion or an insertion in one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209 and Q264. The most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID No. 6 of WO 2006/066594 and residues 36-483 of the SEQ ID No.: 4 are those that have substitutions:

M197T;

H156Y + A181T + N190F + A209V + Q264S; or

G48A + T 49I + G107A + H156Y + A181T + N190F + I201F + A209V + Q264S.

[0347] Additional amylases that are suitable are amylases having SEQ ID No. 6 in WO 99/019467 or variants thereof that have 90% sequence identity with SEQ ID No. 6. Preferred variants of SEQ ID No. 6 are those that have a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269 . Particularly preferred amylases are those that have a removal at positions R181 and G182, or at positions H183 and G184.

[0348] Additional amylases that can be used are those that have SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of wO 96/023873 or variants thereof having 90% sequence identity with SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3 or SEQ ID N .: 7. The preferred variants of SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3 or SEQ ID No.: 7 are those that have a substitution , a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96 / 023873 for numbering. The most preferred variants are those that have a deletion in two selected positions of 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID No.: 1, SEQ ID No.: 2 or SEQ ID No.: 7 are those that have a deletion at positions 183 and 184 and a substitution at one or more of positions 140, 195, 206 , 243, 260, 304 and 476.

[0349] Other amylases that can be used are amylases having SEQ ID No. 2 of WO 08/153815, SEQ ID No. 10 in WO 01/66712 or variants thereof having 90 % sequence identity with SEQ ID No. 2 of WO 08/153815 or 90% sequence identity with SEQ ID No. 10 in WO 01/66712. Preferred variants of SEQ ID No. 10 in WO 01/66712 are those that have a substitution, a deletion or an insertion in one or more of the following positions: 176, 177, 178, 179, 190, 201,207, 211 and 264.

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[0350] Additional suitable amylases are amylases that have SEQ ID No. 2 of WO 09/061380 or variants that have 90% sequence identity with SEQ ID No. 2 of them. Preferred variants of SEQ ID No.: 2 are those that have a C-terminal truncation and / or a substitution, a deletion or an insertion in one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. The most preferred variants of SEQ ID NO: 2 are those that have the substitution in one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T1311, T165I, K178L, T182G, M201L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E and G475K and / or disposal in position R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those that have the substitutions:

N128C + K178L + T182G + Y305R + G475K;

N128C + K178L + T182G + F202Y + Y305R + D319T + G475K;

S125A + N128C + K178L + T182G + Y305R + G475K; or

S125A + N128C + T1311 + T1651 + K178L + T182G + Y305R + G475K, where the variants are truncated at the C-terminal and optionally further comprise a replacement at position 243 and / or a deletion at position 180 and / or position 181

[0351] Other suitable amylases are the alpha-amylase having SEQ ID No. 12 in WO01 / 66712 or a variant that has at least 90% sequence identity with SEQ ID No. 12. Preferred amylase variants are those that have a substitution, a deletion or an insertion in one or more of the following positions of SEQ ID NO: 12 in WO01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants that have a removal of D183 and G184 and that have the R118K, N195F, R320K and R458K substitutions, and a variant that additionally has the substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferably a variant that additionally has substitutions in all these positions.

[0352] Other examples are variants of amylase such as those described in WO2011 / 098531, WO2013 / 001078 and WO2013 / 001087.

[0353] Commercially available amylases are Duramyl ™, Termamyl ™, Fungamyl ™, Stainzyme ™, Stainzyme Plus ™, Natalase ™, Liquozyme X and BAN ™ (from Novozymes A / S), and Rapidase ™, Purastar ™ / Effectenz ™, Powerase and Preferenz S100 (from Genencor International Inc./DuPont).

Peroxidases / oxidases

[0354] A peroxidase according to the invention is a peroxidase enzyme comprised by the classification of enzymes EC 1.11.1.7, as established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment obtained therefrom. , which exhibits peroxidase activity.

[0355] Suitable peroxidases include those of plant, bacterial or fungal origin. Chemically modified or designed protein mutants are included. Examples of useful peroxidases include Coprinopsis peroxidases, for example, from C. cinerea (EP 179,486), and variants thereof such as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

[0356] A peroxidase according to the invention also includes a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds showing chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze the formation of hypochlorite from chloride ions.

[0357] In one embodiment, the haloperoxidase of the invention is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, that is, a haloperoxidase containing vanadate. In a preferred method of the present invention, the haloperoxidase containing vanadate is combined with a source of chloride ions.

[0358] Haloperoxidases have been isolated from many different fungi, in particular from the dematiaceous hyphomycetes fungal group, such as Caldariomyces, for example, C. fumago, Alternaria, Curvularia, for example, C. verruculosa and C. inaequalis, Drechslera , Ulocladium and Botrytis.

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[0359] Haloperoxidases have also been isolated from bacteria such as Pseudomonas, for example, P. pyrrocinia and Streptomyces, for example, S. aureofaciens.

[0360] In a preferred embodiment, the haloperoxidase is derivable from Curvularia sp., In particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461, or Geniculosporium sp. as described in WO 01/79460.

[0361] An oxidase according to the invention includes, in particular, any laccase enzyme comprised by the classification of enzymes EC 1.10.3.2, or any fragment obtained therefrom that exhibits laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), or a bilirubin oxidase (EC 1.3.3.5).

[0362] Preferred laccase enzymes are enzymes of microbial origin. Enzymes can be obtained from plants, bacteria or fungi (including filamentous fungi and yeasts).

[0363] Suitable examples of fungi include a laccase derivable from a strain of Aspergillus, Neurospora, for example, N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, for example, T. villosa and T Versicolor, Rhizoctonia, for example, R. solani, Coprinopsis, for example, C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, for example, P. condelleana, Panaeolus, for example, P. papilionaceus, Myceliophthora, for example, M. thermophila, Schyitalidium, for example, S. thermophilum, Polyporus, for example, P. pinsitus, Phlebia, for example, P. radiata (WO 92/01046), or Coriolus, for example, C. hirsutus (JP 2238885).

[0364] Suitable examples of bacteria include a laccase derivable from a strain of Bacillus.

[0365] A laccase obtained from Coprinopsis or Myceliophthora is preferred; in particular a laccase obtained from Coprinopsis cinerea, as described in WO 97/08325; or from Myceliophthora thermophila, as described in WO 95/33836.

[0366] The detergent enzyme (s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all these enzymes. A detergent additive of the invention, that is, a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, suspension, etc. Preferred detergent additive formulations are granules, in particular non-powder granules, liquids, in particular stabilized liquids, or suspensions.

[0367] Non-powder granules can be produced, for example, as described in US 4,106,991 and 4,661,452 and can optionally be coated by methods known in the art. Examples of waxy coating materials are products of poly (ethylene oxide) (polyethylene glycol, PEG) with average molar weights of 1000 to 20 000; ethoxylated nonylphenols having 16 to 50 units of ethylene oxide; ethoxylated fatty alcohols where the alcohol contains 12 to 20 carbon atoms and in which there are 15 to 80 units of ethylene oxide; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding a polyol such as propylene glycol, a sugar or sugar alcohol, or acid inhibitors. lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method described in EP 238,216.

Other materials

[0368] Any detergent component known in the art for use in detergents can also be used. Other optional detergent components include anti-corrosive agents, anti-shrinking agents, anti-redeposition agents of dirt, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrating / disintegrating agents, dyes, enzymatic stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric conditioners including clays, fillers / treatment auxiliaries, fluorescent whitening agents / optical brighteners, foam enhancers, foam regulators (soaps), perfumes, dirt suspending agents, softeners, suppressants Soap, tanning inhibitors, and absorption agents, either alone or in combination. Any ingredient known in the art for use in detergents can be used. The choice of such constituents is within the skill of the expert.

Dispersants

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[0369] The detergent compositions of the present invention may also contain dispersants. In particular, powdered detergents may comprise dispersants. Suitable water-soluble organic materials include homo- or copolymeric acids or their salts, wherein the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series, volume 71, Marcel Dekker, Inc.

Dye transfer inhibition agents

[0370] The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, N-vinyl pyrrolidone and N-vinylimidazole copolymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a composition in question, dye transfer inhibiting agents may be present at levels of about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the composition.

Fluorescent whitening agent

[0371] The detergent compositions of the present invention will preferably also contain additional components that can dye the items being cleaned, such as fluorescent bleaching agents or optical brighteners. When present, the brightener is preferably at a level of about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most frequently used fluorescent bleaching agents are those belonging to the classes of derivatives of diaminoestylbeno sulphonic acid, diarylpyrazoline derivatives and bisphenyl diestyryl derivatives. Examples of the type of diamino stilbene sulfonic acid derivative of fluorescent bleaching agents include sodium salts of: 4,4'-bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2 '-disulfonate, 4,4'-bis- (2,4-dianilino-s-triazin-6- ylamino) stilbene-2,2'-disulfonate, 4,4'-bis- (2-anilino-4- ( N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis- (4-phenyl-1,2,3-triazole- 2-yl) stilbene-2,2'-disulfonate and 5- (2H-naphtho [1,2-c (] [1,2,3] triazol-2-yl) -2 - [(E) - 2- Phenylvinyl] sodium benzenesulfonate Preferred fluorescent bleaching agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland Tinopal DMS is the disodium salt of 4,4'-bis- (2-morpholino-4-aniline - s-triazin-6-ylamino) stilbene-2,2'-disulfonate.Tinopal CBS is the disodium salt of 2,2'-bis- (phenyl-styryl) -disulfonate.It is also a preferred fluorescent bleaching agent available commercially Parawhite KX, supplied by Para Mount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is a disodium salt of 4.4'-bis- (sulfoestiryl) -biphenyl also known as disodium disulfonate of diestyrylbiphenyl. Other fluorescent agents suitable for use in the invention include 1-3-diaryl pyrazolines and 7-alkylaminocoumarins.

[0372] Suitable fluorescent brightener levels include levels below about 0.01, 0.05, about 0.1 or even about 0.2% by weight at levels above 0.5 or even 0.75% by weight.

Dirt Release Polymers

[0373] The detergent compositions of the present invention may also include one or more soil release polymers that aid in the removal of dirt from fabrics such as cotton and polyester-based fabrics, in particular the removal of hydrophobic dirt. of polyester-based fabrics. The dirt release polymers can be, for example, nonionic or anionic terephthalate based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of dirt release polymers are alkoxylated fat cleaning amphiphilic polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure as described in detail in Wo 2009/087523 (which is incorporated herein by reference). In addition, random graft copolymers are suitable dirt release polymers. Suitable graft copolymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (which are incorporated herein by reference). Other dirt release polymers are substituted polysaccharide structures, especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003/040279 (which are incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, non-ionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures of

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the same. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxy ethyl cellulose, hydroxylpropyl methyl cellulose, carboxymethyl cellulose ester, and mixtures thereof.

Anti-redeposition agents

[0374] The detergent compositions of the present invention may also include one or more antiredeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), acrylic acid homopolymers, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described under dirt release polymers above can also function as anti-redeposition agents.

Rheology modifiers

[0375] The detergent compositions of the present invention may also include one or more rheology modifiers, structuring or thickeners, other than viscosity reducing agents. Rheology modifiers are selected from the group consisting of non-polymeric crystalline hydroxy functional materials, polymeric rheology modifiers that impart shear thinning characteristics to the matrix of a liquid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example, as shown in EP 2169040.

[0376] Other suitable accompanying materials include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, bactericides, binders, carriers, dyes, enzymatic stabilizers, softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, soap suppressants, solvents, and structuring agents for liquid detergents and / or elasticizing agents of the structure.

Formulation of detergent products

[0377] The detergent composition of the invention may be in any convenient form, for example, a tablet, a homogeneous tablet, a tablet having two or more layers, a bag having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular compact or concentrated liquid.

[0378] The bags can be configured with a single compartment or with multiple compartments. It can be of any shape, shape and material that is suitable for retaining the composition, for example, without allowing the release of the composition of the bag before coming into contact with water. The bag is made of water-soluble film that includes an internal volume. Said internal volume can be divided into compartments of the bag. Preferred films are polymeric materials, preferably polymers that are formed in a film or sheet. Preferred polymers, copolymers or derivatives thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxymethyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, malt dextrin, polymethyl acryloxy ethyl cellulose polymethyl acrylate (HPMC). Preferably, the level of polymer in the film, for example, PVA, is at least about 60%. The preferred average molecular weight will typically be from about 20,000 to about 150,000. The films may also be of mixed compositions comprising mixtures of hydrolytically degradable and water-soluble polymers such as polylactide and polyvinyl alcohol (known under commercial reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticizers such as glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The bags may comprise a cleaning composition for solid laundry or partial components and / or a liquid cleaning composition or partial components separated by the water-soluble film. The compartment for liquid components may have a different composition from that of the compartments containing solids: US2009 / 0011970 A1.

[0379] The detergent ingredients can be physically separated from each other by compartments in water-soluble bags or in different layers of tablets. Therefore, the negative storage interaction between the components can be avoided. Different dissolution profiles of each of the compartments can also result in delayed dissolution of selected components in the wash solution.

[0380] A liquid or gel detergent, which is not unit dose, may be aqueous, and typically contains at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to approximately 55% water, up to approximately 45% water, up to approximately 35% water. Other types of liquids, which include, but are not limited to, alkanols, amines, diols, ethers and polyols, may be included in an aqueous liquid or gel. A liquid or aqueous gel detergent may contain 0-30% organic solvent.

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[0381] A liquid or gel detergent may be non-aqueous. Pills of laundry soap

[0382] The DNase of the invention can be added to laundry soap bars and used for hand wash fabrics, fabrics and / or textiles. The term laundry soap bar includes laundry tablets, soap tablets, combo tablets, synthetic detergent tablets and detergent tablets. The types of tablets usually differ in the type of surfactant they contain, and the term laundry soap tablet includes those that contain fatty acid soaps and / or synthetic soaps. The laundry bar has a physical form that is solid and not a liquid, a gel or a powder at room temperature. The term solid is defined as a physical form that does not change significantly over time, that is, if a solid object (for example, a bar of laundry soap) is placed inside a container, the solid object does not change to fill the container in which it has been placed. The tablet is a solid typically in the form of a bar, but may have other solid forms such as circular or oval.

[0383] The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and / or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerin, pH control compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or a salt of a monovalent cation and a organic anion where the monovalent cation can be, for example, Na +, K + or NH4 + and the organic anion can be, for example, formate, acetate, citrate or lactate so that the salt of a monovalent cation and an organic anion can be, for example, sodium formate.

[0384] The laundry soap bar may also contain complexing agents such as EDTA and HEDP, perfumes and / or different types of fillers, surfactants, for example, anionic synthetic surfactants, adjuvants, polymeric dirt release agents , detergent chelants, stabilizing agents, fillers, dyes, dyes, dye transfer inhibitors, alkoxylated polycarbonates, soap suppressants, structuring agents, binders, leaching agents, bleaching activators, clay dirt removal agents, anti-redeposition agents, agents of polymer dispersion, brighteners, fabric softeners, perfumes and / or other compounds known in the art.

[0385] The laundry soap bar may be processed in a conventional laundry soap tablet manufacturing equipment such as, but not limited to: mixers, compressors, p. eg, a dual phase vacuum compressor, extruders, cutters, logo stammers, cooling tunnels and packers. The invention is not limited to preparing soap bars for laundry by any single method. The premix of the invention can be added to soap at different stages of the process. For example, the premix containing a soap, DNase, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion can be prepared and the mixture is then compressed. DNase and optional additional enzymes can be added at the same time as the protease inhibitor, for example, in liquid form. In addition to the mixing stage and the compression stage, the process can also comprise the milling, extrusion, cutting, stamping, cooling and / or packaging stages.

Enzyme formulation in cogranules

[0386] DNase can be formulated as a granule, for example, as a co-granule that combines one or more enzymes. Each enzyme will then be present in more granules ensuring a more constant distribution of the enzymes in the detergent. This also reduces the physical segregation of different enzymes due to different particle sizes. The methods for producing multienzyme cogranulates for the detergent industry are described in the IP.com disclosure IPCOM000200739D.

[0387] Another example of enzyme formulation through the use of cogranulates is described in WO 2013/188331, which refers to a detergent composition comprising (a) a multi-enzymatic co-granule; (b) less than 10% by weight of zeolite (anhydrous base); and (c) less than 10% by weight of phosphate salt (anhydrous base), wherein said enzymatic co-granule comprises from 10 to 98% by weight of moisture reduction component and the composition additionally comprises from 20 to 80% by weight of detergent moisture reduction component. WO 2013/188331 also refers to a method of treating and / or cleaning a surface, preferably a tissue surface that includes the steps of (i) contacting said surface with the detergent composition as claimed and described herein. in an aqueous wash solution, (ii) rinse and / or dry the surface.

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[0388] The multienzyme co-granule may comprise a DNase and (a) one or more enzymes selected from the group consisting of first wash lipases, cleaning cellulases, xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccase and mixtures thereof; and (b) one or more enzymes selected from the group consisting of hemicellulases, proteases, care cellulases, cellobiose dehydrogenases, xylanases, phospho lipases, esterases, cutinases, pectinases, mannases, pectate lyases, keratinases, reductases, oxidases, phenoxidases, ligninases , pululanasas, tanasas, pentosanasas, lichenase glucanases, arabinosidases, hyaluronidase, chondroitinase, amylases, and mixtures thereof.

[0389] The invention is further summarized in the following paragraphs:

1. Use of a polypeptide that has DNase activity to prevent, reduce or eliminate a biofilm of an article, where the polypeptide is obtained from a fungal source and the article is a textile.

2. Use according to paragraph 1 to prevent, reduce or eliminate the stickiness of the article.

3. Use according to any of paragraphs 1 or 2 to pretreat the stains in the article.

4. Use according to any of paragraphs 1-3 to prevent, reduce or eliminate redeposition of dirt during a wash cycle.

5. Use according to any of paragraphs 1-4 to prevent, reduce or eliminate adhesion of dirt to the article.

6. Use according to any of the preceding paragraphs to maintain or improve the whiteness of the article.

7. Use according to any of the preceding paragraphs, where the polypeptide is the polypeptide of paragraphs 47-56.

8. Use according to any of the preceding paragraphs, where a bad smell is reduced or eliminated from the article.

9. Use according to any of the preceding paragraphs, where the bad smell is caused by E-2-nonenal.

10. Use according to any of the preceding paragraphs, where the amount of E-2-nonenal present in a wet textile is reduced or eliminated.

11. Use according to any of the preceding paragraphs, where the amount of E-2-nonenal present in a dry textile is reduced or eliminated.

12. Detergent composition comprising a polypeptide having deoxyribonuclease (DNase) activity and a detergent adjunct ingredient, wherein the polypeptide is obtained from a fungal source.

13. Detergent composition according to paragraph 12, where the polypeptide is obtained from Aspergillus.

14. Detergent composition according to any of the preceding composition paragraphs, where the polypeptide is obtained from Aspergillus oryzae.

15. Detergent composition according to any of the preceding paragraphs, wherein the polypeptide is the polypeptide of paragraphs 47-56.

16. Detergent composition according to any of the preceding composition paragraphs, wherein the detergent attachment ingredient is selected from the group consisting of surfactants, adjuvants, flocculation aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, Catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay dirt removal / antiredeposition agents, brighteners, soap suppressants, dyes, perfumes, structure elasticizing agents , fabric softeners, carriers, hydrotropes, adjuvants and adjuvants, tissue matting agents, anti-foaming agents, dispersants, processing aids, and / or pigments.

17. Detergent composition according to any of the preceding composition paragraphs, wherein the composition further comprises one or more enzymes selected from the group consisting of proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannases, arabinases, galactanases, xylanases and oxidases

18. Detergent composition according to any of the preceding composition paragraphs, where the enzyme is a protease, which is of animal, plant or microbial origin.

19. Detergent composition according to any of the preceding composition paragraphs, where the protease is chemically modified or protein designed.

20. Detergent composition according to any of the preceding composition paragraphs, wherein the protease is a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.

21. Detergent composition according to any of the preceding composition paragraphs, where the protease is selected from the group consisting of Bacillus, for example, Novo subtilisin, Carlsberg subtilisin, 309 subtilisin, 147 subtilisin 147, subtilisin 168, bovine trypsin, trypsin of porcine origin and Fusarium protease.

22. Detergent composition according to any of the preceding composition paragraphs, where the protease has at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 10.

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23. Detergent composition according to any of the preceding paragraphs, where the protease has at least 90% identity with the amino acid sequence of SEQ ID NO: 10 or a variant thereof with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274, preferably the variant is an alkaline protease that It has at least 90% identity with the amino acid sequence of SEQ ID NO: 10 with the following substitution: M222S or the N76D + G195E substitutions.

24. Detergent composition according to any of the preceding composition paragraphs, wherein the detergent composition is capable of reducing the adhesion of the bacteria selected from the group consisting of Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus , Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp. to a surface, or to release bacteria from a surface to which they adhere.

25. Detergent composition according to any of the preceding composition paragraphs, where the surface is a textile surface.

26. Detergent composition according to any of the preceding composition paragraphs, where the textile is made of cotton, cotton / polyester, polyester, polyamide, polyacryl and / or silk.

27. Detergent composition according to any of the preceding composition paragraphs, where the composition is a tablet, a homogeneous tablet, a tablet that has two or more layers, a bag that has one or more compartments, a regular or compact powder, a granule , a paste, a gel, or a regular compact or concentrated liquid.

28. Detergent composition according to any of the preceding composition paragraphs, wherein the composition is a liquid detergent, a powder detergent or a granulated detergent.

29. A washing method for washing an article comprising the steps of:

to. Exposing an article to a wash solution comprising a polypeptide of paragraphs 47-56 or a detergent composition according to any of paragraphs 12-28;

b. Complete at least one wash cycle; Y

C. Optionally rinse the item,

Where the article is a textile.

30. Method according to paragraph 28, where the pH of the wash solution is in the range of 1 to 11.

31. Method according to any of the preceding method paragraphs, where the pH of the wash solution is in the range of 5.5 to 11, such as in the range of 7 to 9, in the range of 7 to 8 or in the range of 7 to 8.5.

32. Method according to any of the preceding method paragraphs, where the temperature of the wash solution is in the range of 5 ° C to 95 ° C, or in the range of 10 ° C to 80 ° C, in the range of 10 ° C to 70 ° C, in the range of 10 ° C to 60 ° C, in the range of 10 ° C to 50 ° C, in the range of 15 ° C to 40 ° C or in the range of 20 ° C at 30 ° C.

33. Method according to any of the preceding method paragraphs, where the temperature of the wash solution is 30 ° C.

34. Method according to any of the preceding method paragraphs, wherein the method further comprises draining the wash solution or part of the wash solution after the end of a wash cycle.

35. Method according to any of the preceding method paragraphs, where the article is exposed to the wash solution for a first and optionally a second or third wash cycle.

36. Method according to any of the preceding method paragraphs, where the article is rinsed after being exposed to the wash solution.

37. Method according to any of the preceding method paragraphs, where the article is rinsed with water or with water comprising a conditioner.

38. Method according to any of the preceding method paragraphs, where the stickiness of the article is reduced.

39. Method according to any of the preceding method paragraphs, where the stains present in the article are pretreated with a polypeptide of paragraphs 47-56 or a detergent composition according to any of paragraphs 12-28.

40. Method according to any of the preceding method paragraphs, where the redeposition of the dirt is reduced.

41. Method according to any of the preceding method paragraphs, where the adhesion of the dirt to the article is reduced or eliminated.

42. Method according to any of the preceding method paragraphs, where the whiteness of the article is maintained or improved.

43. Method according to any of the preceding method paragraphs, where the bad smell is reduced or eliminated from the article.

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44. Method according to any of the preceding method paragraphs, where the bad smell is caused by E-2-nonenal.

45. Method according to any of the preceding method paragraphs, where the amount of E-2-nonenal present in a wet or dry textile is reduced or eliminated.

46. Method according to any of the preceding method paragraphs, where the concentration of the polypeptide in the wash solution is at least 1 mg of DNase protein, such as at least 5 mg of protein, preferably at least 10 mg of protein. , more preferably of at least 15 mg of protein, even more preferably of at least 20 mg of protein, most preferably of at least 30 mg of protein, and even more preferably of at least 40 mg of protein per liter of wash solution.

47. A polypeptide that has DNase activity, selected from the group consisting of:

to. a polypeptide that has at least 60% sequence identity with the mature polypeptide of SEQ iD #: 2 or a polypeptide that has at least 60% sequence identity with the mature polypeptide of SEQ ID No. : 9;

b. a polypeptide encoded by a polynucleotide that hybridizes under low astringency conditions with

i. the mature polypeptide coding sequence of SEQ ID No. 1,

ii. the cDNA sequence thereof, or

iii. the full length complement of (i) or (ii);

C. a polypeptide encoded by a polynucleotide having at least 60% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof;

d. A variant of the mature polypeptide of SEQ ID No. 2 comprising a substitution, elimination, and / or insertion in one or more positions or a variant of the mature polypeptide of SEQ ID No. 9 comprising a substitution, elimination, and / or insertion in one or more positions; Y

and. A fragment of the polypeptide of (a), (b), (c), or (d) having DNase activity;

48. The polypeptide of paragraph 47, which has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the mature polypeptide of SEQ ID No.: 2 or the mature polypeptide of SEQ ID No. 9.

49. The polypeptide according to paragraph 47 or 48, which is encoded by a polynucleotide that hybridizes under low astringency conditions, low-medium astringency conditions, medium astringency conditions, medium-high astringency conditions, high astringency conditions, or very high astringency conditions with

i. the mature polypeptide coding sequence of SEQ ID No. 1,

ii. the cDNA sequence thereof, or

iii. the complement throughout its length of (i) or (ii).

50. The polypeptide according to any of paragraphs 47-49, which is encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at minus 97%, at least 98%, at least 99% or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

51. The polypeptide according to any of paragraphs 47-50, which comprises or consists of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2.

52. The polypeptide according to any of paragraphs 47-50, which comprises or consists of SEQ ID No. 9 or the mature polypeptide of SEQ ID No. 9.

53. The polypeptide according to paragraph 51, wherein the mature polypeptide is amino acids 1 to 206 of SEQ ID NO: 2.

54. The polypeptide according to paragraph 52, wherein the mature polypeptide is amino acids 1 to 204 of SEQ ID NO: 9.

55. The polypeptide according to any of paragraphs 47-56, which is a variant of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, elimination, and / or insertion in one or more positions or a variant of the polypeptide mature of SEQ ID #: 9 comprising a substitution, elimination, and / or insertion in one or more positions.

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56. The polypeptide according to paragraph 55, which is a fragment of SEQ ID No. 2, where the fragment has DNase activity or a fragment of SEQ ID No. 9, where the fragment has DNase activity .

57. A polynucleotide encoding the polypeptide according to any of paragraphs 47-56.

58. A nucleic acid or expression vector construct comprising the polynucleotide of paragraph 57 operably linked to one or more control sequences that direct the production of the polypeptide into an expression host.

59. A recombinant host cell comprising the polynucleotide of paragraph 57-58 operatively linked to one or more control sequences that direct the production of the polypeptide.

60. A method for producing the polypeptide of any of paragraphs 47-56, comprising culturing a cell, which in its wild-type form produces the polypeptide, under conditions conducive to the production of the polypeptide.

61. The method of paragraph 60, further comprising recovering the polypeptide.

62. A method for producing a DNase activity polypeptide, comprising culturing the host cell of paragraph 59 under conditions conducive to the production of the polypeptide.

63. The method of paragraph 62, further comprising recovering the polypeptide.

64. A polynucleotide encoding a signal peptide that comprises or consists of amino acids -37 to -1 of SEQ ID NO: 2.

65. The polynucleotide according to claim 64, further comprising a polynucleotide encoding a propeptide comprising or consisting of amino acids -15 to -1 of SEQ iD No.: 2

66. A nucleic acid or expression vector construct comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 65, where the gene is foreign to the polynucleotide encoding the signal peptide.

67. A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 65, wherein the gene is foreign to the polynucleotide encoding the signal peptide.

68. A method of producing a protein, comprising culturing a recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 64, where the gene is foreign to the polynucleotide encoding the signal peptide, under auspicious conditions. for protein production.

69. The method of paragraph 67, which further includes the recovery of the protein.

70. An isolated polynucleotide encoding a propeptide that comprises or consists of amino acids -15 to -1 of SEQ ID NO: 2.

71. A nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 64, where the gene is foreign to the polynucleotide encoding the propeptide.

72. A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 64, wherein the gene is foreign to the polynucleotide encoding the propeptide.

73. A method of producing a protein, comprising the culture of the recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph 62, where the gene is foreign to the polynucleotide encoding the propeptide, under conditions conducive to protein production

74. The method of paragraph 73, which further includes the recovery of the protein.

75. A complete broth formulation or cell culture composition comprising a polypeptide of any of paragraphs 47-56.

76. Article washed according to the method of any of paragraphs 29-46.

77. A pharmaceutical composition comprising a polypeptide having DNase activity and a pharmaceutical adjunct ingredient, wherein the polypeptide is obtained from a fungal source.

78. Pharmaceutical composition according to paragraph 77, where the polypeptide having DNase activity is obtained from Aspergillus.

79. Pharmaceutical composition according to any of paragraphs 77-78, where the polypeptide having DNase activity is obtained from Aspergillus oryzae.

80. Pharmaceutical composition according to any of paragraphs 77-79, wherein the polypeptide is the polypeptide of paragraphs 47-56.

81. Pharmaceutical composition according to any of paragraphs 77-80, wherein the composition is formulated as a toothpaste, a liquid toothpaste, a mouthwash, a tablet or a gingival massage ointment.

82. Pharmaceutical composition according to any of paragraphs 77-81, further comprising one or more of an antimicrobial compound, such as an antibacterial compound, an antiparasitic compound, an antifungal compound and an antiviral compound.

83. A permanent medical device characterized by the fact that at least a portion of a surface that comes into contact with the patient of said device is coated with the pharmaceutical composition of any of paragraphs 77-83.

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84. The device according to paragraph 83 where said device is a catheter such as a central venous catheter, intravascular catheter, urinary catheter, Hickman catheter, peritoneal dialysis catheter, endrotracheal catheter, or where the device is a mechanical heart valve, a cardiac pacemaker, an arteriovenous connector, a sclera closure, a joint prosthesis, a tympanostomy tube, a tracheotomy tube, a voice prosthesis, a penile prosthesis, an artificial urinary sphincter, a synthetic pubovaginal sling, a surgical suture, a bone anchor, a bone screw, an intraocular lens, a contact lens, an intrauterine device, an aortofemoral graft, a vascular graft, a needle, a Luer-Lok connector, a needleless connector or a surgical instrument.

85. A method of producing the polypeptide of any of paragraphs 47-56, comprising culturing the host cell of paragraph 59 under conditions conducive to the production of the polypeptide.

86. The method of paragraph 85, further comprising recovering the polypeptide.

87. The recombinant host cell of paragraph 59 further comprising a polynucleotide encoding a second polypeptide of interest; preferably an enzyme of interest; more preferably a secreted enzyme of interest; even more preferably a hydrolase, isomerase, ligase, lyase, oxidoreductase, or a transferase; and most preferably the secreted enzyme is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitin, cutinase , cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucan-transferase, glucoamylase, invertase, lacasa, lipase, mannosidase, mutanasa, oxidase, pectinolytic enzyme, peroxidase, phytase, polypheoloxidase, ribase protease enzyme xylanase

88. The recombinant host cell of paragraph 87, wherein the second polypeptide of interest is heterologous or homologous to the host cell.

89. The recombinant host cell of paragraph 87 or 88, which is a fungal host cell; preferably a filamentous fungal host cell; more preferably a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Pacilotorotomyotomyotomyotomyotoma, Pylorus, Pyloromycesus, Pylorus, Pylorus, Pylorus, Pylorus, Pylorus, Pyramid , Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma; most preferably a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Ceriporiopsis Cerpispsis Ceripus Ceriporiopsis subvermispora, inops Chrysosporium, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum , Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fus arium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes trichoderma, Trichodermata trichorumummata, Trichoderma trichitema, Trichoderma trichormata, Trichoderma trichormata , or Trichoderma viride.

90. The recombinant host cell of paragraph 87 or 88, which is a bacterial host cell; preferably a prokaryotic host cell; more preferably a gram-positive host cell; even more preferably a host cell of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces; and most preferably a host cell of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licillifususususususususususususususususususus, Bacillus subtilis, and Bacillus thuringiensis.

91. A method for producing the second polypeptide of interest as defined in any of paragraphs 87-88, which comprises culturing the host cell of any of paragraphs 87-90 under conditions conducive to the production of the second polypeptide of interest.

92. The method of paragraph 91, further comprising recovering the second polypeptide of interest.

93. Detergent composition according to any of the preceding composition paragraphs, wherein the detergent attached ingredient is a surfactant.

94. Detergent composition according to any of the preceding composition paragraphs, wherein the detergent attached ingredient is an adjuvant.

95. Detergent composition according to any of the preceding composition paragraphs, where the attached detergent ingredient is a clay dirt removal / antiredeposition agent.

96. Detergent composition according to paragraphs 28, wherein the composition is a liquid detergent composition, comprising a surfactant and a detergent adjuvant in a total concentration of at least

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3% by weight, and a microcapsule containing detergent enzyme, where the membrane of the microcapsule is produced by crosslinking a polyaminated polyamine with a molecular weight of more than 1 kDa.

97. Detergent composition according to paragraphs 96, wherein the reactive amino groups of the polyamramified polyamine constitute at least 15% of the molecular weight.

98. Detergent composition according to any of paragraphs 96-97, wherein the microcapsule is produced using an acid chloride as a crosslinking agent.

99. Detergent composition according to any of paragraphs 96-98, where the diameter of the microcapsule is at least 50 microns or more.

100. Detergent composition according to any of paragraphs 96-99, wherein the microcapsule contains at least 1% by weight of active enzyme.

101. Detergent composition according to any of paragraphs 96-100, which also includes an alcohol, such as a polyol.

102. Detergent composition according to any of paragraphs 96-101, wherein the surfactant is an anionic surfactant.

103. Detergent composition according to any of paragraphs 96-102, which is a composition for liquid laundry.

104. Detergent composition according to any of paragraphs 96-103, containing less than 90% by weight of water.

105. Detergent composition according to any of paragraphs 96-104, wherein the detergent enzyme is a polypeptide having DNase, protease, amylase, lipase, cellulase, mannanase, pectinase, or oxidoreductase activity.

106. Detergent composition according to any of paragraphs 96-105, wherein the protease is a metalloprotease or an alkaline serine protease, such as a subtilisin.

107. Detergent composition according to any of paragraphs 96-105, wherein the polypeptide having DNase activity is the polypeptide according to any of claims 47-54.

108. Detergent composition according to any of paragraphs 96-107, wherein the microcapsule is produced by interfacial polymerization using an acid chloride as a crosslinking agent.

109. Detergent composition according to any of paragraphs 96-108, wherein the polyamramified polyamine is a polyethyleneimine.

110. Detergent composition according to any of paragraphs 96-109, wherein the microcapsule comprises a source of Mg2 +, Ca2 +, or Zn2 + ions, such as a sparingly soluble salt of Mg2 +, Ca2 +, or Zn2 +.

Detergent tests and compositions

Detergent compositions

[0390] The detergent composition mentioned below can be used in combination with the enzyme of the invention.

Biotex Black (liquid)

[0391] 5-15% of anionic surfactants, <5% of non-ionic surfactants, perfume, enzymes, DMDM and hydantoin. Composition of Ariel Sensitive White & Color, liquid detergent composition

[0392] Water, ethoxy sulfate alcohol, ethoxylated alcohol, amino oxide, citric acid, C12-18 crowned palm kernel fatty acid, protease, glycosidase, amylase, ethanol, 1.2 propanediol, sodium formate, calcium chloride , sodium hydroxide, silicone emulsion, trans-sulfated EHDQ (the ingredients are listed in descending order).

Detergent composition model WFK IEC-A (powder)

[0393] Ingredients: 8.8% linear sodium alkylbenzene sulphonate, 4.7% C12-18 ethoxylated fatty alcohol (7 EO), 3.2% sodium soap, 3.9% DC2- antifoam 4248S, 28.3% zeolite 4A sodium aluminum silicate, 11.6% sodium carbonate, 2.4% sodium salt of a copolymer of maleic and acrylic acid (Sokalan CP5), 3.0% sodium silicate, 1.2% carboxymethylcellulose, 2.8% Dequest 2066, 0.2% optical bleach, 6.5% sodium sulfate, 0.4% protease.

Detergent composition model A (liquid)

[0394] Ingredients: 12% of LAS, 11% of AEO Biosoft N25-7 (NI), 7% of AEOS (SLES), 6% of MPG (monopropylene glycol), 3% of ethanol, 3% of TEA, 2, 75% cocoa soap, 2.75% soy soap, 2% glycerol,

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2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTMPA and 0.2% PCA (all percentages are p / p)

Composition of Ariel Actilift (liquid)

[0395] Ingredients: 5-15% anionic surfactants; <5% non-ionic surfactants, phosphonates, soap; enzymes, optical brighteners, benzisothiazolinone, methylisothiazolinone, perfumes, alpha-isomethyl ionone, citronellol, geraniol, linalool.

Composition of Ariel Actilift Color & Style (liquid)

[0396] Ingredients: 5-15% anionic surfactants; <5% non-ionic surfactants, phosphonates, soap; enzymes, perfumes, benzisothiazolinone, methylisothiazolinone, alpha-isomethyl ionone, butylphenyl methylpropional, citronellol, geraniol, linalool.

Composition of Persil Small & Mighty (liquid)

[0397] Ingredients: 15-30% anionic surfactants, non-ionic surfactants, 5-15% soap, <5% polycarboxylates, perfume, phosphates, optical brighteners

Persil 2 in 1 with Comfort Passion Flower Powder

[0398] Sodium sulfate, sodium carbonate, sodium dodecylbenzenesulfonate, bentonite, sodium carbonate peroxide, sodium silicate, zeolite, water, citric acid, TAED, C12-15 Pareth-7, stearic acid, perfume, acrylic acid Sodium / MA copolymer, cellulose gum, modified corn starch, sodium chloride, tetrasodium etidronate, EDTMP sodium calcium, anilinomomorpholinotriacinylamino stylbenosulfonate disodium, sodium bicarbonate, phenylpropyl ethyl methicone, butylphenyl methylpropional stearyrate, stearate carbonate calcium, sodium polyacrylate, alpha-isomethyl ionone, disodium diestyrylbiphenyl disulphonate, cellulose, protease, limonene, PEG-75, titanium dioxide, dextrin, sucrose, sodium polyaryl sulphonate, Cl 12490, Cl 45100, Cl 42090, thiosulfate sodium, Cl 61585.

Persil biological powder

[0399] Sucrose, sorbitol, aluminum silicate, polyoxymethylene melamine, sodium polyaryl sulfonate, Cl 61585, Cl 45100, lipase, amylase, xanthan gum, hydroxypropylmethylcellulose, Cl 12490, diestyrylbiphenyl disulfonate disodium, sodium thiosulfate, 42090, sodium 420 , Cl 11680, ethidronic acid, Tetrasodium EDTA.

Persil biological tablets

[0400] Sodium carbonate, sodium carbonate peroxide, sodium bicarbonate, zeolite, water, sodium silicate, sodium lauryl sulfate, cellulose, TAED, sodium dodecylbenzenesulfonate, hemicellulose, lignin, lauryl glycoside, sodium acrylic acid / MA copolymer, bentonite, sodium chloride, perfume, tetrasodium etidronate, sodium sulfate, sodium polyacrylate, dimethicone, disodium anilinomorpholinotriazinylamino stylbenosulfonate, calcium dodecylbenzene sulfonic acid, trimethylsiloxysilicate, calcium carbonate, cellulose, titanium dioxide, PEG-75 titanium dioxide , protease, modified corn starch, sucrose, Cl 12490, sodium polyaryl sulphonate, sodium thiosulfate, amylase, kaolin,

Persil Color Care Organic Powder

[0401] Subtilisin, imidazolidinone, hexyl cinnamal, sucrose, sorbitol, aluminum silicate, polyoxymethylene melamine, Cl 61585, Cl 45100, lipase, amylase, xanthan gum, hydroxypropylmethylcellulose, Cl 12490, diestyrylbiphenyl disodium disodium disodium 420 , mannanase, Cl 11680, etidronic acid, Tetrasodium EDTA.

Persil Color Care biological tablets

[0402] Sodium bicarbonate, sodium carbonate, zeolite, water, sodium silicate, sodium lauryl sulfate, cellulose gum, sodium dodecylbenzenesulfonate, lauryl glycoside, sodium chloride, sodium acrylic acid / MA copolymer, perfume, thioglycolate sodium, PVP, sodium sulfate, tetrasodium etidronate, sodium polyacrylate, dimethicone, bentonite, dodecylbenzene sulfonic acid, trimethylsiloxysilicate, calcium carbonate, cellulose, PEG-75, titanium dioxide, dextrin, protease, modified corn starch, sucrose, sodium thiosulfate, amylase, Cl 74160, kaolin.

Persil Dual Action Capsules Bio

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[0403] MEA-dodecylbenzenesulfonate, MEA-hydrogenated cocoate, C12-15 Pareth-7, dipropylene glycol, water, tetrasodium etidronate, polyvinyl alcohol, glycerin, aziridine, ethoxylated homopolymer, propylene glycol, perfume, sodium pentamethylene phosphonate diethylenetriamine, , MEA-sulfate, ethanolamine, subtilisin, glycol, butylphenyl methylpropional, boronic acid, (4-formylphenyl), hexyl cinnamal, limonene, linalool, diestyrylbiphenyl disulfonate disodium, alpha-isomethyl ionone, geraniol, amylase, yellow dye polymeric polymeric, talc, sodium chloride, benzisothiazolinone, mannanase, denatonium benzoate.

Persil 2 in 1 with Comfort Sunshiny Days Powder

[0404] Sodium sulfate, sodium carbonate, sodium dodecylbenzenesulfonate, bentonite, sodium carbonate peroxide, sodium silicate, zeolite, water, citric acid, TAED, C12-15 Pareth-7, perfume, stearic acid, acrylic acid Sodium / MA copolymer, cellulose gum, modified corn starch, sodium chloride, tetrasodium etidronate, EDTMP sodium calcium, anilinomorpholinotriazinylamino stylbenosulfonate disodium, sodium bicarbonate, phenylpropyl ethyl methicone, butylphenyl methylpropional stearate, stearate carbonyl calcium, sodium polyacrylate, geraniol, disodium diestyrylbiphenyl disulfonate, cellulose, protease, PEG-75, titanium dioxide, dextrin, sucrose, sodium polyaryl sulphonate, Cl 12490, Cl 45100, Cl 42090, sodium thiosulfate, Cl 61585.

Persil Small & Mighty 2 in 1 with Comfort Sunshiny Days

[0405] Water, C12-15 Pareth-7, sodium dodecylbenzenesulfonate, propylene glycol, hydrogenated sodium cocoate, triethanolamine, glycerin, hydrogenated TEA cocoate, perfume, sodium chloride, polyquaternium-10, PVP, polymeric pink dye, sulfate sodium, disodium diethyrylbiphenyl disulfonate, methylpropional butylphenyl, styrene / acrylate copolymer, cinnamonyl hexyl, citronellol, eugenol, polyvinyl alcohol, sodium acetate, isopropyl alcohol, polymeric yellow dye, sodium lauryl sulfate.

Persil Small & Mighty Bio

[0406] Water, MEA-dodecylbenzenesulfonate, propylene glycol, sodium laureth sulfate, C12-15 Pareth-7, hydrogenated TEA cocoate, MEA-citrate, ethoxylated aziridine homopolymer, MEA-etidronate, triethanolamine, perfume, acrylate copolymer, sorbol , MEA-sulfate, sodium sulphite, disodium diestyrylbiphenyl disulfonate, methylpropional butylphenyl, styrene / acrylate copolymer, citronellol, sodium sulfate, peptides, salts, fermentation sugars (process), subtilisin, glycerin, boronic acid, (4- formylphenyl), geraniol, pectate lyase, amylase, sodium lauryl sulfate, mannanase, Cl 42051.

Persil Small & Mighty Capsules Biological

[0407] MEA-dodecylbenzenesulfonate, MEA-hydrogenated cocoate, C12-15 Pareth-7, dipropylene glycol, water, glycerin, polyvinyl alcohol, perfume, ethoxylated aziridine homopolymer, diethylenetriamine pentamethylene phosphonate sodium, propylene glycol, sorbitol, M-sulfate, M-sulfate, MIT ethanolamine, subtilisin, glycol, methylpropional butylphenyl, cinnamal hexyl, starch, boronic acid, (4-formylphenyl), limonene, disodium linalool, diethyrylbiphenyl disulfonate, alpha-isomethyl ionone, geraniol, amylase, talcum, polymeric sodium chloride , benzisothiazolinone, denatonium benzoate, polymeric yellow dye, mannanase.

Persil Small & Mighty Capsules Color Care

[0408] MEA-dodecylbenzenesulfonate, MEA-hydrogenated cocoate, C12-15 Pareth-7, dipropylene glycol, water, glycerin, polyvinyl alcohol, perfume, ethoxylated aziridine homopolymer, diethylenetriamine pentamethylene phosphonate sodium, propylene glycol, MEA-sulfate, MEA-sulfate PVP, sorbitol, methylpropional butylphenyl, subtilisin, cinnamal hexyl, starch, limonene, linalool, boronic acid, (4-formylphenyl), alpha-isomethyl ionone, geraniol, talc, polymeric blue dye, denatonium benzoate, polymeric yellow dye.

Persil Small & Mighty Color Care

[0409] Water, MEA-dodecylbenzenesulfonate, propylene glycol, sodium laureth sulfate, C12-15 Pareth-7, hydrogenated TEA cocoate, MEA-citrate, ethoxylated aziridine homopolymer, MEA-etidronate, triethanolamine, perfume, acrylate copolymer, sorbol , MEA-sulfate, sodium sulphite, glycerin, methylpropional butylphenyl, citronellol, sodium sulfate, peptides, salts, fermentation sugars (process), styrene / acrylate copolymer, subtilisin, boronic acid, (4- formylphenyl), geraniol, lycta pectate, amylase, sodium lauryl sulfate, mannanase, Cl 61585, Cl 45100.

Composition of Fairy Non Bio (liquid)

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[0410] Ingredients: 15-30% anionic surfactants, 5-15% non-ionic surfactants, soap, benzisothiazolinone, methylisothiazolinone, perfumes

Detergent composition model T (powder)

[0411] Ingredients: 11% of LAS, 2% of AS / AEOS, 2% of soap, 3% of AEO, 15.15% of sodium carbonate, 3% of sodium silicate, 18.75% of zeolite, 0.15% chelator, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP (all percentages are w / w).

Model X detergent composition (powder)

[0412] Ingredients: 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% sokalan, 35.5% sodium sulfate (all percentages are p / p).

Composition of Ariel Actilift Color & Style (powder)

[0413] Ingredients: 15-30% anionic surfactants, <5% non-ionic surfactants, phosphonates, polycarboxylates, zeolites; enzymes, perfumes, cinnamal hexyl.

Composition of Ariel Actilift (powder)

[0414] Ingredients: 5-15% anionic surfactants, oxygen-based bleaching agents, <5% non-ionic surfactants, phosphonates, polycarboxylates, zeolites, optical brighteners, enzymes, perfumes, methylpropional butylphenyl, coumarin, cinnamal hexyl

Composition of Persil Megaperls (powder)

[0415] Ingredients: 15-30% of the following: anionic surfactants, oxygen bleaching agent and zeolites, less than 5% of the following: nonionic surfactants, phosphonates, polycarboxylates, soap. Additional ingredients: perfumes, cinnamal hexyl, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol.

Gain Liquid, Original:

[0416] Ingredients: water, ethoxy sulfate alcohol, diethylene glycol, ethoxylated alcohol, ethanolamine, linear alkylbenzene sulphonate, sodium fatty acids, ethoxylated polyethyleneimine, citric acid, borax, sodium cumene sulfonate, propylene glycol, DTPA, diathylethyl disodium dipulphonate tetramine, sodium hydroxide, sodium formate, calcium formate, dimethicone, amylase, protease, Liquitint ™, hydrogenated castor oil, fragrance

Tide Liquid, Original:

[0417] Ingredients: linear alkylbenzene sulphonate, propylene glycol, citric acid, sodium hydroxide, borax, ethanolamine, ethanol, alcohol sulfate, ethoxylated polyethyleneimine, sodium fatty acids, diquaternium ethoxy sulfate, protease, diethylene glycol, laureth-9, oxide of alkyldimethylamine, fragrance, amylase, disodium diamino stilbene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquitint ™ Blue, dimethicone.

Liquid Tide, Free and Gentle:

[0418] Water, sodium alcoholetoxy sulfate, propylene glycol, borax, ethanol, linear sodium alkylbenzene sulphonate, salt, ethoxylated polyethyleneimine, diethylene glycol, sulfated and ethoxylated hexamethylene diamine, ethoxylated alcohol, linear alkylbenzene sulfate, sodium salt, MEA salt, sodium salt Sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diamino stilbene, disulfonate, amylase, protease, dimethicone, benzisothiazolinone.

Tide Coldwater Liquid, Fresh Scent:

[0419] Water, alcoholetoxy sulfate, linear alkylbenzene sulphonate, diethylene glycol, propylene glycol, ethanolamine, citric acid, borax, alcohol sulfate, sodium hydroxide, polyethyleneimine, ethoxylate, sodium fatty acids, ethanol, protease, Laureth-9, ethoxy sulfate diquaternium, lauramine oxide, sodium cumene, sulfonate, fragrance, DTPA, amylase, disodium diamino stilbene, disulfonate, sodium formate, diethyrylbiphenyl disulfonate disodium, calcium formate, polyethylene glycol 4000, mannanase, pectinase, Liquitint ™ Blue, Liquitone ™ Blue, Liquitone ™ Blue, Liquitone ™, Blue

Tide TOTALCARE ™ Liquid, Cool Cotton:

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[0420] Water, alcoholetoxy sulfate, propylene glycol, sodium fatty acids, laurtrimony chloride, ethanol, sodium hydroxide, cumene sodium sulfonate, citric acid, ethanolamine, diethylene glycol, silicone polyether, borax, fragrance, ethoxylated polyethyleneimine, protease, Laureth-9.

[0421] DTPA, quaternium polyacrylamide chloride, disodium diamino stilbene disulfonate, sodium formate, Liquitint ™ Orange, dipropylethyl tetramine, dimethicone, cellulase.

Liquid Tide Plus Bleach Alternative ™, Vivid White and Bright, Original and Clean Breeze:

[0422] Water, sodium alcoholetoxy sulfate, sodium alkyl sulfate, MEA citrate, linear alkylbenzene sulphonate, MEA salt, propylene glycol, diethylene glycol, ethoxylated polyethyleneimine, ethanol, sodium fatty acids, ethanolamine, lauramine oxide, borax, Laureth -9, DTPA, sodium cumene sulphonate, sodium formate, calcium formate, linear alkylbenzene sulphonate, sodium salt, alcohol sulfate, sodium hydroxide, diquaternium ethoxy sulfate, fragrance, amylase, protease, mannanase, pectinase, diamino stilbene disulfonate of disodium, benzisothiazolinone, Liquitint ™ Blue, dimethicone, dipropylethyl tetramine.

Liquid Tide HE, Original Scent:

[0423] Water, sodium alcoholetoxy sulfate, MEA citrate, sodium alkyl sulfate, ethoxylated alcohol, linear alkylbenzene sulphonate, MEA salt, sodium fatty acids, ethoxylated polyethyleneimine, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, boraxin, polyethylene Propoxylated ethoxylate, ethanol, sodium cumene sulphonate, fragrance, DTPA, disodium diamino stilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, lauramine oxide, Liquitint ™ Blue, dimethicone / polydimethyl silicone.

Tide TOTALCARE HE Liquid, renewing Rain:

[0424] Water, alcoholetoxy sulfate, linear alkylbenzene sulphonate, ethoxylated alcohol, citric acid, ethanolamine, sodium fatty acids, diethylene glycol, propylene glycol, sodium hydroxide, borax, ethoxylated polyethyleneimine, silicone polyether, ethanol, protease, sodium cumene sulfonate , diquaternium ethoxy sulfate, Laureth-9, fragrance, amylase, DTPA, disodium diamino stilbene disodium, diestyrylbiphenyl disodium disodium, sodium formate, calcium formate, mannanase, Liquitint ™ Orange, dimethicone, quaternium diphenyl tetramyl cellulose, quaternium diphenyl chloride, .

Tide liquid HE Free:

[0425] Water, alcoholetoxy sulfate, diethylene glycol, monoethanolamine citrate, sodium formate, propylene glycol, linear alkylbenzene sulphonates, ethanolamine, ethanol, ethoxylated polyethyleneimine, amylase, benzisothiazoline, borax, calcium formate, calcium diethyl acetate citric acid dimethicone, diquaternium ethoxy sulfate, disodium diamino stilbene disulfonate, Laureth-9, mannanase, protease, sodium cumene sulphonate, sodium fatty acids.

Tide Coldwater HE Liquid, Fresh Scent:

[0426] Water, alcoholetoxy sulfate, MEA citrate, alcohol sulfate, ethoxylated alcohol, linear alkyl benzene sulphonate MEA, sodium fatty acids, ethoxylated polyethyleneimine, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, borax, polyethyleneimine ethoxydane ethane, ethoxydane ethane, ethoxydane ethane, ethoxydane ethane Sodium, fragrance, DTPA, disodium diaminostilbene disulphonate MEA, protease, mannanase, cellulase, amylase, sodium formate, calcium formate, lauramine oxide, Liquitint ™ Blue, dimethicone.

Tide for Coldwater HE Free Liquid:

[0427] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate: sodium salt, ethoxylated alcohol, linear alkylbenzene sulphonate: MEA salt, sodium fatty acids, ethoxylated polyethyleneimine, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, bicarbonate , protease, propoxylated ethoxylated polyethyleneimine, ethanol, sodium cumene sulphonate, amylase, citric acid, DTPA, disodium diamino stilbene disulfonate, sodium formate, calcium formate, dimethicone.

Tide Simply Clean & Fresh:

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[0428] Water, ethoxylated alcohol sulfate, linear alkylbenzene sulphonate, sodium / MEA salts, propylene glycol, diethylene glycol, sodium formate, ethanol, borax, sodium fatty acids, fragrance, lauramine oxide, DTPA, ethoxylated polyethylene amine, Calcium formate, disodium diamino stilbene disulfonate, dimethicone, tetramine, Liquitint ™ Blue.

Tide Pods, Ocean Mist, Mystic Forest, Spring Meadow:

[0429] Linear alkylbenzene sulphonates, C12-16 Pareth-9, propylene glycol, alkoxyloxy sulfate, water, ethoxylated polyethyleneimine, glycerin, fatty acid salts, polyvinyl acetate PEG-136, ethylene diamine disuccinic salt, sodium ethanolamine monohydrate , diethylenetriamine sodium pentaacetate, diestyrylbiphenyl disulfonate disodium, calcium formate, mannanase, exyloglucanase, sodium formate, hydrogenated castor oil, natalase, dyes, thermamyl, subtilisin, benzisothiazoline, perfume.

Tide to Go:

[0430] Deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, dimethyl alkyl amine oxide, citric acid, sodium hydroxide, trimethoxy benzoic acid, frangacia.

Tide Stain Release Liquid:

[0431] Water, ethoxylated alkyl, linear alkylbenzenesulfonate, hydrogen peroxide, diquaternium ethoxy sulfate, ethanolamine, diethyrylbiphenyl disodium disulfonate, tetrabutyl ethylidinobisphenol, F&DC Yellow 3, fragrance.

Tide Stain Release Powder:

[0432] Sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxy benzene sulfonate, sodium polyacrylate, water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodium palmitate, amylase, protease, modified starch, FD&C Blue 1, fragrance.

Tide Stain Release, Pre Treater Spray:

[0433] Water, ethoxylated alkyl, MEA borate, linear alkylbenzenesulfonate, propylene glycol, diquaternium ethoxy sulfate, calcium chloride enzyme, protease, ethanolamine, benzoisothiazolinone, amylase, sodium citrate, sodium hydroxide, fragrance.

Tide to Go Stain Eraser:

[0434] Water, alkyl amine oxide, phenyl dipropylene glycol ether, hydrogen peroxide, citric acid, sodium salt of ethylene diamine disuccinic acid, sodium alkyl sulfate, fragrance.

Tide boost with Oxi:

[0435] Sodium bicarbonate, sodium carbonate, sodium percarbonate, ethoxylated alcohol, sodium chloride, maleic / acrylic copolymer, nonanoyloxy benzene sulfonate, sodium sulfate, dye, diethylenetriamine pentaacetate sodium salt, hydrated aluminosilicate (zeolite), polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate, starch, water, fragrance.

Tide Stain Release boost Duo Pac:

[0436] Polyvinyl alcohol bag film, where a liquid part and a powder part are packaged: Liquid ingredients: dipropylene glycol, diquaternium ethoxy sulfate, water, glycerin, LiquitintTM Orange, powdered ingredients: sodium percarbonate, nonanoyloxy benzene sulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzenesulfonate, maleic / acrylic copolymer, water, amylase, polyethylene glycol, sodium palmitate, modified starch, protease, glycerin, DTPA, fragrance.

Tide Ultra Stain Release:

[0437] Water, sodium alcoholetoxy sulfate, linear alkylbenzene sulphonate, sodium / MEA salts, MEA citrate, propylene glycol, ethoxylated polyethyleneimine, ethanol, diethylene glycol, propoxyethoxylated polyethyleneimine, sodium fatty acids, protease, sodium borax, cumene sulfonate DTPA, fragrance, amylase, disodium diamino stilbene disulfonate, calcium formate, sodium formate, gluconase, dimethicone, Liquitint ™ Blue, mannanase.

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Ultra Tide with a Touch of Downy® Powdered Detergent, April Fresh / Clean Breeze / April Essence:

[0438] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicate, alkyl sulfate, nonanoyloxybenzenesulfonate, DTPA, polyethylene glycol 4000, silicone, ethoxylate, fragrance, oxide of polyethylene, palmitic acid, disodium diamino stilbene disulfonate, protease, Liquitint ™ Red, FD&C 1 Blue, cellulase.

Ultra Tide with a Touch of Downy Clean Breeze:

[0439] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate: sodium / MEA salts, propylene glycol, ethoxylated polyethyleneimine, ethanol, diethylene glycol, polyethyleneimine, propoxyethoxylate, diquaternium ethoxysulfate, alcohol sulfate, dimethyl oxaxamide, dimethyl sulphonate, alcohol sulfate, dimethoxy ethoxylate , sodium fatty acids, DTPA, protease, sodium bisulfite, disodium diamino stilbene disulfonate, amylase, gluconase, castor oil, calcium formate, MEA, styrene acrylate copolymer, sodium formate, Liquitint ™ Blue.

Ultra Tide with Downy Sun Blossom:

[0440] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate: sodium / MEA salts, propylene glycol, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, ethoxylated polyethyleneimine, alcohol sulfate, dimethicone, fragrance, borax acids, sodium acids , DTPA, protease, sodium bisulfite, disodium diamino stilbene disulfonate, amylase, castor oil, calcium formate, MEA, styrene acrylate copolymer, propanaminium propanamide, gluconase, sodium formate, Liquitint ™ Blue.

Ultra Tide with Downy April Fresh / Sweet Dreams:

[0441] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate: sodium / MEA salts, propylene glycol, ethoxylated polyethyleneimine, ethanol, diethylene glycol, propoxyethoxylated polyethyleneimine, diquaternium ethoxysulfate, alcohol sulfate, dimethoxyrate, alcohol, Sodium fatty acids, DTPA, protease, sodium bisulfite, disodium diamino stilbene disulfonate, amylase, gluconase, castor oil, calcium formate, MEA, styrene acrylate copolymer, propanaminium propanamide, sodium formate, Liquitint ™ Blue.

Ultra Tide Free Powdered Detergent:

[0442] Sodium carbonate, sodium aluminosilicate, alkyl sulfate, sodium sulfate, linear alkylbenzene sulphonate, water, sodium polyacrylate, silicate, ethoxylate, sodium percarbonate, polyethylene glycol 4000, protease, diamino stilbene disulfonate disodium, silicone, cellulase.

Ultra Tide Powdered Detergent, Clean Breeze / Spring Lavender / mountain Spring:

[0443] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, diamino stilodium disulfonate palmitic acid, protease, silicone, cellulase.

Ultra Tide HE (High Efficiency) Powdered Detergent, Clean Breeze:

[0444] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, water, nonanoyloxybenzenesulfonate, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, diamino stylbeno sulfide of disodium, protease, silicone, cellulase.

Ultra Tide Coldwater Powdered Detergent, Fresh Scent:

[0445] Sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfate, linear alkylbenzene sulphonate, water, nonanoyloxybenzenesulfonate, sodium polyacrylate, silicate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, natalase, palmitic acid, Protease, disodium diamino stilbene disulfonate, FD&C 1 Blue, silicone, cellulase, alkyl ether sulfate.

Ultra Tide with bleach Powdered Detergent, Clean Breeze:

[0446] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000,

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fragrance, DTPA, palmitic acid, protease, disodium diamino-stilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate.

Ultra Tide with Febreeze Freshness ™ Powdered Detergent, Spring Renewal

[0447] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, protease, diaminoestyl disodium disulfonate, silicone, FD&C Blue 1.

Liquid Tide Plus with Febreeze Freshness - Sport HE Active Fresh:

[0448] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate, sodium salt, linear alkylbenzene sulphonate: MEA salt, ethoxylated alcohol, sodium fatty acids, propylene glycol, diethylene glycol, ethoxylated ethoxylated polyethyleneimine, diquaxysulfate diqua ethanol, sodium cumene sulphonate, borax, fragrance, DTPA, sodium bisulfate, disodium diamino stilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, lauramine oxide, Liquitint ™ Blue, dimethicone / polydimethyl silicone.

Tide Plus Febreeze Freshness Spring & Renewal:

[0449] Water, sodium alcoholetoxy sulfate, linear alkylbenzene sulphonate: sodium / MEA salts, MEA citrate, propylene glycol, ethoxylated polyethyleneimine, fragrance, ethanol, diethylene glycol, propoxyethoxylated polyethyleneimine, protease, alcohol sulfate, borax acids, , DTPA, disodium diamino stilbene disulfonate, MEA, mannanase, gluconase, sodium formate, dimethicone, Liquitint ™ Blue, tetramine.

Liquid Tide Plus with Febreeze Freshness, Sport HE Victory Fresh:

[0450] Water, sodium alcoholetoxy sulfate, MEA citrate, linear alkylbenzene sulphonate, sodium salt, linear alkylbenzene sulphonate: MEA salt, ethoxylated alcohol, sodium fatty acids, propylene glycol, diethylene glycol, ethoxylated ethoxylated polyethyleneimine, diquaxysulfate diqua ethanol, sodium cumene sulphonate, borax, fragrance, DTPA, sodium bisulfate, disodium diamino stilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, lauramine oxide, Liquitint ™ Blue, dimethicone / polydimethyl silicone.

Tide Vivid White + Bright Powder, Original:

[0451] Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulphonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, protease, disodium diamino stilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate.

HEY SPORT TEX WASH Detergent

[0452] Water, dodecylbenzenesulfonate, laureth-11, lanolin peg-75, propylene glycol, bare alcohol., Potassium soyate, potassium hydroxide, disodium cocoanfodiacetate, perfume, zinc co-alkaline ethoxyamine triacetate, perfume, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol.

[0453] Products called Tide, Ariel, Gain and Fairy are commercially available products supplied by Procter & Gamble. Products called Persil are commercially available products supplied by Unilever and Henkel. Products called Hey Sport are commercially available products supplied by Hey Sport.

 Ingredient

 Quantity (in% by weight)

 Anionic detersive surfactant (such as alkylbenzene sulphonate, ethoxylated alkyl sulfate and mixtures thereof

 from 8% by weight to 15% by weight

 Nonionic detersive surfactant (such as alkyl ethoxylated alcohol)

 from 0.5% by weight to 4% by weight

 Cationic detersive surfactant (such as quaternary ammonium compounds)

 from 0% to 4% by weight

 Other detersive surfactant (such as zwitterionic detersive surfactants,

 from 0% by weight to 4 by weight

 amphoteric surfactants and mixtures thereof)

 %

 Carboxylate polymer (such as copolymers of maleic acid and acrylic acid)

 from 1% by weight to 4% by weight

 Polyethylene glycol polymer (such as a polyethylene glycol polymer comprising polyvinyl acetate side chains)

 from 0.5% by weight to 4% by weight

 Polyester dirt release polymer (such as Repel-o-tex and / or Texcare polymers)

 0.1 to 2% by weight

 Cellulosic polymer (such as carboxymethyl cellulose, methyl cellulose and combinations thereof)

 from 0.5% by weight to 2% by weight

 Other polymer (such as amine polymers, dye transfer inhibitor polymers, hexamethylene diamine derived polymers, and mixtures thereof)

 from 0% by weight to 4% by weight

 Zeolite adjuvant and phosphate adjuvant (such as zeolite 4A and / or sodium tripolyphosphate)

 from 0% by weight to 4% by weight

 Other adjuvant (such as sodium citrate and / or citric acid)

 from 0% by weight to 3% by weight

 Carbonate salt (such as sodium carbonate and / or sodium bicarbonate)

 from 15% by weight to 30% by weight

 Silicate salt (such as sodium silicate)

 from 0% by weight to 10% by weight

 Filler (such as sodium sulfate and / or bio-fillers)

 from 10% by weight to 40% by weight

 Available oxygen source (such as sodium percarbonate)

 from 10% by weight to 20% by weight

 Bleach activator (such as tetraacetylethylene diamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)

 from 2% by weight to 8% by weight

 Bleaching catalyst (such as oxaziridinium-based bleaching catalyst and / or transition metal bleaching catalyst)

 from 0% by weight to 0.1% by weight

 Other bleach (such as performated reducing and / or peracid bleach)

 from 0% by weight to 10% by weight

 Chelator (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or diphosphonic hydroxyethane acid (HEDP)

 from 0.2% by weight to 1% by weight

 Photobleach (such as sulfonated zinc and / or aluminum phthalocyanine)

 from 0% by weight to 0.1% by weight

 Matting agent (such as direct violet 99, acid red 52, acid blue 80, direct

 from 0% by weight to 1 weight

 violet 9, solvent violet 13 and any combination thereof)

 %

 Brightener (such as brightener 15 and / or brightener 49)

 from 0.1% by weight to 0.4% by weight

 Protease (such as Savinase, Savinase Ultra, Purafect, FN3, FN4 and any combination thereof)

 from 0.1% by weight to 0.4% by weight

 Amylase (such as Termamyl, Termamyl Ultra Natalase, Optisize, Stainzyme, Stainzyme Plus, and any combination thereof)

 from 0.05% by weight to 0.2% by weight

 Cellulase (such as Carezyme and / or Celluclean)

 from 0.05% by weight to 0.2% by weight

 Lipase (such as Lipex, Lipolex, Lipoclean and any combination thereof)

 from 0.2% to 1% by weight

 Other enzyme (such as xyloglucanase, cutinase, lycta pectate, mannanase, bleaching enzyme)

 from 0% by weight to 2% by weight

 Fabric softener (such as montmorillonite and / or polydimethylsiloxane clay (PDMS)

 from 0% by weight to 4% by weight

 Flocculant (such as polyethylene oxide)

 from 0% by weight to 1% by weight

 Foam suppressor (such as silicone and / or fatty acid)

 from 0% by weight to 0.1% by weight

 Perfume (such as perfume microcapsule, spray perfume, starch encapsulated perfume chords, perfume-loaded zeolite, and any combination thereof)

 from 0.1% by weight to 1% by weight

 Aesthetics (such as colored soap rings and / or colored spots / streaks)

 from 0% by weight to 1% by weight

 Miscellany

 The rest

 Ingredient

 Quantity

 Polymer containing the carboxyl group (comprising from about 60% to about 70% by mass of an acrylic acid based monomer (A); and

 from about 0.5% by weight to

 from about 30% to about 40%) by mass of a monomer containing the sulfonic acid group (B); and where the average molecular weight is from about 23,000 to about 50,000 preferably in the range of about 25,000 to about 38,000 as described in WO2014032269.

 approximately 1.5% by weight

 Amylase (Stainzyme Plus (R), which has an enzyme activity of 14 mg of active enzyme / g

 from about 0.1% by weight to about 0.5% by weight

 Anionic detersive surfactant (such as alkyl benzene sulfonate, ethoxylated alkyl sulfate and mixtures thereof)

 from about 8% by weight to about 15% by weight

 Nonionic detersive surfactant (such as ethoxylated alcohol alkyl)

 from about 0.5% by weight to 4% by weight

 Cationic detersive surfactant (such as quaternary ammonium compounds)

 from about 0% by weight to about 4% by weight

 Other detersive surfactant (such as zwitterionic detersive surfactants, amphoteric surfactants and mixtures thereof)

 from about 0% by weight to 4% by weight

 Carboxylate polymer (such as copolymers of maleic acid and acrylic acid)

 from about 1% by weight to about 4% by weight

 Polyethylene glycol polymer (such as a polyethylene glycol polymer comprising polyvinyl acetate side chains)

 from about 0% by weight to about 4% by weight

 Polyester dirt release polymer (such as Repel-O-Tex (R) and / or Texcare (R) polymers)

 from about 0.1% by weight to about 2% by weight

 Cellulosic polymer (such as carboxymethyl cellulose, methyl cellulose and combinations thereof)

 from about 0.5% by weight to about 2% by weight

 Other polymer (such as amine polymers, dye transfer inhibitor polymers, hexamethylene diamine derived polymers, and mixtures thereof)

 from about 0% by weight to about 4% by weight

 Zeolite adjuvant and phosphate adjuvant (such as zeolite 4A and / or sodium tripolyphosphate)

 from about 0% by weight to about 4% by weight

 Other adjuvant (such as sodium citrate and / or citric acid)

 from about 0% by weight to about 3% by weight

 Carbonate salt (such as sodium carbonate and / or sodium bicarbonate)

 from about 15% by weight to about 30% by weight

 Silicate salt (such as sodium silicate)

 from about 0% by weight to about 10% by weight

 Stuffing (such as sodium sulfate and / or bio-filled)

 from about 10% by weight to about 40% by weight

 Available oxygen source (such as sodium percarbonate)

 from about 10% by weight to about 20% by weight

 Bleach activator (such as tetraacetylethylene diamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)

 from about 2% by weight to about 8% by weight

 Bleaching catalyst (such as oxaziridinium-based bleaching catalyst and / or transition metal bleaching catalyst)

 from about 0% by weight to about 0.1% by weight

 Other bleach (such as preformed reducing and / or peracid bleach)

 from about 0% by weight to about 10% by weight

 Chelator (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or diphosphonic hydroxyethane acid (HEDP)

 from about 0.2% by weight to about 1% by weight

 Photobleach (such as sulfonated zinc and / or aluminum phthalocyanine)

 from about 0% by weight to about 0.1% by weight

 Matting agent (such as direct violet 99, acid red 52, acid blue 80, direct violet 9, solvent violet 13 and any combination thereof)

 from about 0% by weight to about 0.5% by weight

 Brightener (such as brightener 15 and / or brightener 49)

 from about 0.1% by weight to about 0.4% by weight

 Protease (such as Savinase, Polarzyme, Purafect, FN3, FN4 and any combination thereof, typically with an enzymatic activity of about 20 mg to about 100 mg of active enzyme / g)

 from about 0.1% by weight to about 1.5% by weight

 Amylase (such as Termamyl (R), Termamyl Ultra (R), Natalase (R), Optisize HT Plus (R), Powerase (R), Stainzyme (R) and any combination thereof, typically with an enzymatic activity of approximately 10 mg to about 50 mg of active enzyme / g)

 from about 0.05% by weight to about 0.2% by weight

 Cellulase (such as Carezyme (R), Celluzyme (R) and / or Celluclean (R), typically with an enzymatic activity of approximately 10 to 50 mg of active enzyme / g)

 from about 0.05% by weight to 0.5% by weight

 Lipase (such as Lipex (R), Lipolex (R), Lipoclean (R) and any combination thereof, typically with an enzymatic activity of about 10 mg to about 50 mg of active enzyme / g)

 from about 0.2% by weight to about 1% by weight

 Another enzyme (such as xyloglucanase (eg, Whitezyme (R)), cutinase, pectate lyase, mannanase, bleaching enzyme, typically with an enzymatic activity of about 10 mg to about 50 mg of active enzyme / g)

 from 0% by weight to 2% by weight

 Fabric softener (such as montmorillonite and / or polydimethylsiloxane clay (PDMS))

 from 0% by weight to 15% by weight

 Flocculant (such as polyethylene oxide)

 from 0% by weight to 1% by weight

 Soap suppressant (such as silicone and / or fatty acid)

 from 0% by weight to 0.1% by weight

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 Perfume (such as perfume microcapsule, spray perfume, starch encapsulated perfume chords, perfume-loaded zeolite, and any combination thereof)

 from 0.1% by weight to 1% by weight

 Aesthetics (such as colored soap rings and / or colored spots / streaks)

 from 0% by weight to 1% by weight

 Miscellany

 The rest

[0454] All enzyme levels are expressed as crude active enzyme protein per 100 g of detergent composition. Surfactant ingredients can be obtained from BASF, Ludwigshafen, Germany (Lutensol (R)); Shell Chemicals, London, United Kingdom; Stepan, Northfield, III, USA; Huntsman, Huntsman, Salt Lake City, Utah, USA; Clariant, Sulzbach, Germany (Praepagen (R)).

[0455] Sodium tripolyphosphate can be obtained from Rhodia, Paris, France. Zeolite can be obtained from Industrial Zeolite (UK) Ltd, Grays, Essex, UK. Citric acid and sodium citrate can be obtained from Jungbunzlauer, Basel, Switzerland. NOBS is sodium nonanoyloxybenzenesulfonate, supplied by Eastman, Batesville, Ark., USA.

[0456] TAED is tetraacetylethylenediamine, supplied under the Peractive (R) brand by Clariant GmbH, Sulzbach, Germany.

[0457] Sodium carbonate and sodium bicarbonate can be obtained from Solvay, Brussels, Belgium.

[0458] Polyacrylate, polyacrylate / maleate copolymers can be obtained from BASF, Ludwigshafen, Germany.

[0459] Repel-O-Tex (R) can be obtained from Rhodia, Paris, France.

[0460] Texcare (R) can be obtained from Clariant, Sulzbach, Germany. Sodium percarbonate and sodium carbonate can be obtained from Solvay, Houston, Tex., USA.

[0461] The Na salt of ethylenediamine-N, N'-disuccinic acid, isomer (S, S) (EDDS) was supplied by Octel, Ellesmere Port, United Kingdom.

[0462] Hydroxyethane di phosphonate (HEDP) was supplied by Dow Chemical, Midland, Mich., USA.

[0463] Savinase (R), Savinase (R) Ultra, Stainzyme (R) Plus, Lipex (R), Lipolex (R), Lipoclean (R), Celluclean (R), Carezyme (R) enzymes can be obtained Natalase (R), Stainzyme (R), Stainzyme (R) Plus, Termamyl (R), Termamyl (R) ultra, and Mannaway (R) from Novozymes, Bagsvaerd, Denmark.

[0464] Purafect (R), FN3, FN4 and Optisize enzymes can be obtained from Genencor International Inc., Palo Alto, California, USA.

[0465] Direct violet 9 and 99 can be obtained from BASF DE, Ludwigshafen, Germany. Solvent violet 13 can be obtained from Ningbo Lixing Chemical Co., Ltd. Ningbo, Zhejiang, China. Brighteners can be obtained from Ciba Specialty Chemicals, Basel, Switzerland.

[0466] All percentages and proportions are calculated by weight unless otherwise indicated. All percentages and proportions are calculated based on the total composition unless otherwise indicated. It should be understood that each maximum numerical limitation given throughout this specification includes each lower numerical limitation, as if such lower numerical limitations were expressly written in this document. Each minimum numerical limitation given throughout this specification shall include each higher numerical limitation, as if such higher numerical limitations were expressly written in this document. Each numerical range given throughout this specification will include each smaller numerical range that falls within such a wider numerical range, as if such smaller numerical ranges were all expressly written in this document.

Washing tests

Model wash system with Launder-O-Meter (LOM)

[0467] The Launder-O-Meter (LOM) is a medium scale model washing system that can be applied to evaluate up to 20 different washing conditions simultaneously. An LOM is basically a large temperature controlled water bath with 20 closed metal beakers that rotate inside it. Each beaker constitutes a small washing machine and during an experiment, each will contain a solution of a system

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specific detergent / enzyme to be evaluated along with dirty and clean tissues on which it is evaluated. Mechanical tension is achieved by rotating the beakers in the water bath and by including metal balls in the beaker.

[0468] The model washing system with LOM is mainly used in the mid-scale evaluation of detergents and enzymes under European washing conditions. In an experiment with LOM, factors such as the proportion of ballast and dirt and the proportion of tissue and wash solution can be varied. Therefore, the LOM provides the connection between small-scale experiments, such as AMSA and mini-washing, and full-scale experiments that consume more time in front-loading washers.

Model wash system with Mini Launder-O-Meter (MiniLOM)

[0469] MiniLOM is a modified mini washing system from Launder-O-Meter (LOM), which is a medium-scale model washing system that can be applied to evaluate up to 20 different washing conditions simultaneously. An LOM is basically a large temperature controlled water bath with 20 closed metal beakers that rotate inside it. Each beaker constitutes a small washing machine and during an experiment, each will contain a solution of a specific detergent / enzyme system to be evaluated along with the dirty and clean tissues on which it is evaluated. Mechanical tension is achieved by rotating the beakers in the water bath and by including metal balls in the beaker.

[0470] The model washing system with LOM is mainly used in the evaluation of detergents and enzymes at medium scale under European washing conditions. In an experiment with LOM, factors such as the proportion of ballast and dirt and the proportion of tissue and wash solution can be varied. Therefore, the LOM provides the connection between small-scale experiments, such as AMSA and mini-washing, and full-scale experiments that consume more time in front-loading washers.

[0471] In the miniLOM, washings are performed in 50 ml test tubes placed in a Stuart rotator.

Wash test with Terg-O-timeter (TOM)

[0472] The Tergo-To-Meter (TOM) is a medium scale model washing system that can be applied to evaluate 12 different washing conditions simultaneously. A TOM is basically a large temperature controlled water bath with up to 12 open metal beakers submerged in it. Each beaker constitutes a small top-loading style washer and during an experiment, each of them will contain a solution of a specific detergent / enzyme system and the dirty and clean tissues on which its performance is evaluated. Mechanical tension is achieved by rotating a stirring arm, which agitates the liquid inside each beaker. Because the TOM beakers do not have a lid, it is possible to withdraw samples during an experiment with TOM and assess the information online during washing.

[0473] The model washing system with TOM is mainly used in the mid-scale evaluation of detergents and enzymes under US wash conditions. or LA / AP. In an experiment with TOM, factors such as the proportion of ballast and dirt and the proportion of tissue and washing solution can be varied. Therefore, the TOM provides the connection between small-scale experiments, such as AMSA and mini-washing, and full-scale experiments that consume more time in top-loading washers.

[0474] Equipment: the water bath with 12 steel beakers and 1 rotating arm per beaker with a capacity of 500 or 1200 mL of detergent solution. The temperature varies from 5 to 80 ° C. The water bath must be filled with deionized water. The rotation speed can be set at 70 to 120 r.p.m./min.

[0475] Set the temperature in the Terg-O-Tometer and start the rotation in the water bath. Wait for the temperature to adjust (the tolerance is +/- 0.5 ° C). All beakers should be clean and without traces of previous test materials.

[0476] The wash solution with the desired amount of detergent, temperature and water hardness is prepared in a bucket. The detergent is allowed to dissolve during magnetic stirring for 10 min. The wash solution should be used 30 to 60 min after preparation.

[0477] 800 ml wash solution is added in a beaker of the TOM. The wash solution is stirred at 120 rpm. and optionally one or more enzymes are added to the beaker. The samples are sprayed on the beaker and then the ballast load. Time measurement begins when samples and ballast are added to the beaker. The samples are washed for 20 minutes after which stirring is completed. The

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Washing load is subsequently transferred from the TOM beaker to a sieve and rinsed with cold tap water. Dirt samples are separated from the ballast load. The dirt samples are transferred to a 5 L beaker with cold tap water under running water for 5 minutes. The ballast load is maintained separately for the next inactivation. Water is gently removed from the samples by hand and placed on a tray covered with paper. Other paper is placed on top of the samples. The samples are allowed to dry overnight before subjecting the samples to analysis, such as measuring the intensity of color using a Color Eye as described herein.

Enzymatic assays

Test I: DNase activity evaluation

[0478] DNase activity was determined on DNase test agar with methyl green (BD, Franklin Lakes, NJ, USA), which was prepared according to the supplier's manual. Briefly, 21 g of agar was dissolved in 500 ml of water and then autoclaved for 15 min at 121 ° C. The autoclaved agar was warmed at 48 ° C in the water bath, and 20 ml of agar was poured into Petri dishes and allowed to solidify by incubation overnight at room temperature. In solidified agar plates, 5 | _il of enzyme solutions are added, and DNase activity is observed as colorless areas around the stained enzyme solutions.

Essay II

[0479] Analysis of E-2-nonenal in textile using an electronic nose.

[0480] One way to assess the presence of bad smell in tissues is to use E-2-nonenal as a marker for bad smell, since this compound contributes to the bad smell in laundry.

[0481] Add a solution of E-2-nonenal to a 5 cm x 5 cm textile sample and place the sample in a 20 mL glass bottle for GC analysis and cover the bottle. Analyze 5 mL of the empty space of the bottles covered in an electronic nose Heracle II of Alpha MOS, France (double column gas chromatograph with 2 FlD, column 1: MXT5 and column 2: MXT1701) after 20 minutes of incubation at 40 ° C.

Examples

Methods

[0482] General methods of PCR, cloning, nucleotide ligation, etc. they are well known to one skilled in the art and can be found, for example, in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.); "Current protocols in Molecular Biology", John Wiley and children, (1995); Harwood, C. R., and Cutting, S. M. (eds.); "DNA Cloning: A Practical Approach, volumes I and II", D.N. Glover ed. (1985); "Oligonucleotide Synthesis", M.J. Gait ed. (1984); "Nucleic Acid Hybridization", B.D. Hames & S.J. Higgins eds (1985); "A Practical Guide To Molecular Cloning", B. Perbal, (1984).

Example 1

PCR amplification

[0483] All amplifications by PCR were performed in a volume of 100 microL containing 2.5 units of Taq polymerase, 100 ng of pSO2, 250 nM of each dNTP, and 10 pmol of each of the two primers described above in a reaction buffer of 50 mM KCl, 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2.

[0484] The amplification was performed on a Perkin-Elmer Cetus Thermal DNA 480, and consisted of a 3 minute cycle at 94 ° C, followed by 25 cycles of 1 minute at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C.

T ransformation of Aspergillus

[0485] Aspergillus oryzae NBRC4177: available from Institute for fermentation, Osaka; 17-25 Juso, Hammachi 2-Chome Yodogawa-Ku, Osaka, Japan.

[0486] The transformation of Aspergillus was done as described in Christensen et al .; Biotechnology 1988 6 14191422. In summary, A. oryzae mycelia were grown in a nutrient-rich broth. The mycelia were separated from the broth by filtration. The Novozyme® enzyme preparation (Novozymes) was added to the mycelia in the buffer

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osmotically stabilizing such as 1.2 M MgSO4 buffered to pH 5.0 with sodium phosphate. The suspension was incubated for 60 minutes at 37 degrees C with stirring. The protoplast was filtered through mira-cloth to remove mycelial debris. The protoplast was collected and washed twice with STC (1.2 M sorbitol, 10 mM CaCl 2, 10 mM Tris-HCl pH 7.5). The protoplasts were finally resuspended in 200-1000 microl of STC.

[0487] For transformation, 5 pg of DNA was added to 100 pl of protoplast suspension and then 200 pl of PEG solution (60% PEG 4000, 10 mM CaCl2, 10 mM Tris-HCl pH 7 were added) , 5) and the mixture was incubated for 20 minutes at room temperature. The protoplast was collected and washed twice with 1.2 M sorbitol. The protoplast was finally resuspended in 200 μl of 1.2 M sorbitol. Transformants containing the amdS gene were selected for their ability to use acetamide as the sole source of nitrogen in minimal plates (Cove DJ 1966. Biochem. Biophys. Acta. 113: 51-56) containing 1.0 M sucrose as a carbon source, 10 mM acetamide as a nitrogen source. After 4-5 days of cultivation at 37 degrees C, stable transformants appeared as colonies that grow and sporulate vigorously. The transformants were purified twice through conidiospores on the plates of the previous selection.

Example 2

Construction of the Aspergillus pJaL1368 expression cassette

[0488] For the expression of an A. oryzae gene (SEQ ID NO: 1) that encodes a putative nuclease (SEQ ID NO: 2), the intron-containing coding region (SEQ ID NO: 1) was amplified as a 931 bp PCR fragment by primer set oJaL316 (SEQ ID #: 3) and oJaL317 (SEQ ID No.: 4) using A. oryzae nBrC4177 genomic DNA as a model. The 931 bp PCR fragment was digested with BamHI and XhoI resulting in a 919 bp fragment (SEQ ID NO: 5). The 919 BamHI-XhoI fragment was cloned into the corresponding sites in pJaL1262, giving plasmid pJaL1368.

Example 3

Putative A. oryzae nuclease gene expression in the Aspergillus oryzae strain MT3568

[0489] Aspergillus pJaL1368 expression plasmid was transformed into Aspergillus oryzae MT3568 strains as described in Christensen et al .; Biotechnology 1988 6 1419-1422. Twelve randomly selected transformants were purified and inoculated into tubes containing 10 ml of YPM medium (2 g / l yeast extract, 2 g / l peptone, and 2% maltose), which were incubated at 30 ° C , with stirring (200 rpm) for 4 days. The transformants that produced the A. oryzae A0090701000540 protein were identified by the appearance of an extra band at approximately 25 kD (calculated molecular weight 23893 Da) compared to the supernatant of a non-transformed parental strain. The identities of the band were confirmed by 1) the determination of the N-terminus by Edman degradation and 2) the digestion of InGel and MS / MS. N-terminal determination gave two dominant N-terminal sequences ALKTGSG (SEQ ID No.: 6) and KTGSGDS (SEQ ID No.: 7). The MS / MS data further confirm that the putative gene (SEQ ID No.: 1) encodes mature proteins of SEQ ID No. 8 and SEQ ID No. 9, which is part of the protein in SEQ ID NO: 2. The following examples use the Aspergillus oryzae DNase obtained from the aforementioned Aspergillus oryzae MT3568 strains transformed with pJaL1368.

Example 4

Aspergillus oryzae DNase yield (SEQ ID No.: 2) in model detergents and commercial detergents Isolation of bacterial strains specific to laundry

[0490] A strain of Brevundimonas sp. Isolated from the laundry was used in the present example.

[0491] Brevundimonas sp. It was isolated during a study, where bacterial diversity was investigated in the laundry after washing at 15, 40 and 60 ° C, respectively. The study was conducted in laundry collection of Danish homes. For each wash, 20 g of laundry items (dishcloth, towel, dishcloth, bib, shirt tobacco, shirt collar, socks) were used in the range 4: 3: 2: 2: 1: 1: one. Washing was performed in a Laundr-O-Meter (LOM) at 15, 40 or 60 ° C. For washing at 15 and 40 ° C, Ariel Sensitive White & Color was used, while the WFK IEC-A * model detergent was used for washing at 60 ° C. Ariel Sensitive White & Color was prepared weighing 5.1 g and adding tap water up to 1000 ml followed by stirring for 5 minutes. The detergent model WFK IEC-A * (which is available from WFK Testgewebe GmbH) was prepared by weighing 5 g and adding tap water up to 1300 ml followed by stirring for 15 min. Washing was carried out for 1 hour at 15, 40 and 60 ° C, respectively, followed by rinsing 2 times with tap water for 20 min at 15 ° C.

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[0492] The laundry was sampled immediately after washing at 15, 40 and 60 ° C, respectively. Twenty grams of laundry was added to 0.9% (w / v) NaCl (1.06404; Merck, Damstadt, Germany) with 0.5% (w / w) of tween 80 to produce a 1:10 dilution in a bag from Stomacher. The mixture was homogenized using a Stomacher for 2 minutes at medium speed. After homogenization, tenfold dilutions were prepared in 0.9% (w / v) NaCl. The bacteria were listed on tryptone soy agar (TSA) (CM0129, Oxoid, Basingstoke, Hampshire, UK) incubated aerobically at 30 ° C for 5-7 days. To suppress the growth of yeast and molds, 0.2% of sorbic acid (359769; Sigma) and 0.1% of cycloheximide (18079; Sigma) was added. Bacterial colonies were selected from counting plates and purified by scratching twice in TSA. For long-term storage, purified isolates were stored at -80 ° C in TSB containing 20% (w / v) glycerol (49779; Sigma).

Preparation of donor samples with biofilm

[0493] La Brevundimonas sp. precultured in tryptone soy agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, United Kingdom) for 2-5 days at 30 ° C from a single colony, a complete loop was transferred at 10 mL of TSB and incubated for 1 day at 30 ° C with shaking (240 rpm). After propagation, Brevundimonas sp. it was granulated by centrifugation (Sigma Laboratory Centrifuge 6K15) (3000 g at 21 ° C in 7 min) and resuspended in 10 mL of TSB diluted twice with water. The optical density (OD) at 600 nm was measured using a spectrophotometer (POLARstar Omega (BMG Labtech, Ortenberg, Germany) .The fresh TSB diluted twice with water was inoculated for up to an OD600nm of 0.03, and 1.6 mL a 12-well flat bottom polystyrene microplate (3512; Corning Incorporated, Corning, NY, USA) was added to each well, where a round sample (2 cm diameter) of WFK30A sterile polyester was placed. After incubation (24 h at 15 ° C with stirring (100 rpm), the samples were rinsed twice with 0.9% (w / v) NaCl.

Washing experiment

[0494] Five samples rinsed with Brevundimonas sp. they were mixed with five samples of sterile WFK30A polyester in a 50 mL test tube and 10 mL of detergent wash solution comprising the following detergent composition at the mentioned concentrations was added: model A detergent (EU, 3.3 g / L), Ariel Actilift (EU, 6.9 g / L), Persil Small & Mighty (EU, 4 g / L), Fairy (EU, 5.6 g / L) and Original Tide (US, 3 , 2 g / L), detergent model T (EU, 5.3 g / L), detergent model X (D&E, 1.8 g / L), Ariel (EU, 5.3 g / L) and Persil Megaperls ( EU, 4.0 g / L) were added together with 0.7 g / L of dirt (Pigmentschmutz, 09V, wfk, Krefeld, Germany) and Aspergillus oryzae DNase (0.04 ppm) which were found as positive DNase in DNase test agar with methyl green (Test I). The test tubes were placed in a Stuart rotator (Mini LOM) for 1 hour at 30 ° C. The samples were rinsed twice with tap water and dried on filter paper overnight. As controls, washings with detergents mentioned and without the addition of DNase from A. oryzae were done in parallel. The color difference (L values) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted.

[0495] When previously referring to EU conditions, for example (EU, 3.3 g / L), water with hardness of 15 ° dH was used (Ca: Mg: NaHCO3 4: 1: 1.5) . For US detergents (USA, 3.2 g / L), water with hardness of 6 ° dH (Ca: Mg: NaHCOs 2: 1: 1.5) was used. For D&E detergents (developing and emerging markets) (D&E, 1.8 g / L), water with a hardness of 14 ° dH (Ca: Mg: NaHCOs2: 1: 1.5) was used.

[0496] The present example shows that the DNase of A. oryzae prevents the deposition of dirt (antiredeposition) in pre-cultured polyester samples with bacteria (donors) as well as in sterile polyester samples (tracers) added to the wash. The prevention of dirt deposition was observed both in liquid detergents with pH 8.0, and also in powder detergents with pH 10 and Tide Pods. The observed effect is due to the deep cleaning effects of the DNase of A. oryzae (SEQ ID NO: 2). More importantly, the present example shows that the DNase of A. oryzae will prevent the transfer of dirt between different textile items in a wash and thus allow the dirty laundry to be washed with less dirty laundry. This ensures that the whiteness of the textile is improved.

Table 1

 Detergent

 Region pH Color difference (AL)

 Donor Sample Tracer Sample

 Liquids: Model A detergent

 EU 7.7 5.6 5.1

 Ariel Actilift

 EU 8.3 6.9 3.5

 OMO Small & Mighty

 EU 7.9 5.8 3.2

 Fairy

 EU 8.1 5.6 3.5

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 Original Tide

 USA 8.3 5.2 4.8

 Gain

 USA nd 3.8 0.11

 Powder:

 Model T detergent

 EU 10.4 8.9 2.6

 Model X detergent

 D&E 10.2 7.0 4.5

 Ariel Actilift

 EU 10.2 5.6 1.2

 Persil Megaperls

 EU 8.7 4.0 0.4

 Pods:

 Tide Pods

 USA nd 5.7 1.0

 Nd: not determined

Example 5

Aspergillus oryzae DNase yield (SEQ ID No.: 2) in different types of textiles

[0497] To investigate the effects of deep cleaning and bleaching of DNase of A. oryzae on various types of tissues, Brevundimonas sp. in round samples (2 cm diameter) of cotton (WFK10A), polyester / cotton (WKF20A), polyester (wFk30A), polyamide (WFK40A), polyacryl (WFK50A) and silk (WFk70) for 1 day (see example 4 - preparation of donor samples).

[0498] Five samples rinsed with Brevundimonas sp. mixed with five sterile samples in a 50 mL test tube and 10 mL of model A detergent from the model A detergent wash solution (EU, 3.3 g / L) containing 0.7 g / L was added of dirt (Pigmentschmutz, 09V, wfk, Krefeld, Germany) and DNase of Aspergillus oryzae (0.04 ppm). The test tubes were placed in a Stuart rotator (MiniLOM) for 1 hour at 30 ° C. The samples were rinsed twice with tap water and dried on filter paper overnight. As controls, washes were made without the addition of A. oryzae DNase in parallel. The color difference (L values) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted. The present example shows that the A. oryzae DNase prevents the deposition of dirt (anti-redeposition) in various types of textiles and that The whiteness of the textile is improved when the DNase of A. oryzae is present.

Table 2

 Type of textile

 Color difference (AL)

 Cotton (WFK10A)

 3.5

 Cotton / Polyester (WFK20A)

 5.4

 Polyester (WFK30A)

 7.5

 Polyamide (WFK40A)

 3.3

 Polyacryl (WFK50A)

 7.2

 Silk (WFK70A)

 4,5

Example 6

Stability of the Aspergillus oryzae DNase in model A detergent

[0499] To investigate the stability of the detergent and DNase protease of A. oryzae, 20 g of model A detergent was weighed into a 50 ml test tube and 1.35% (w / w) protease 1 was added and protease 2, respectively. In addition, 2 g / l and 4 g / l of protease inhibitor system 1 or 2 g / l of protease inhibitor system 2 were added.

[0500] After incubation at -18 ° C and 37 ° C, respectively, the effects of deep cleansing were investigated in samples of Brevundimonas sp. As controls, model A detergent was used with and without the addition of A. oryzae DNase before washing. The color difference (L values) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted. The present example shows that the A. oryzae DNase is stable in liquid detergent as well as in liquid detergent with several proteases and added protease inhibitor systems.

[0501] Protease 1 used in this experiment is SEQ ID No. 10 with the following modification M222S. Protease 2 is SEQ ID No. 10 with the following modification: N76D + G195E. Protease 1 inhibitor system is 4-formyl-phenyl-boronic acid as described in patent application WO 1996/041859. The protease 2 inhibitor system is the compound Cbz-Gly-Ala-Tyr-H as described in patent application WO 2009/118375.

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 Detergent composition with enzyme mixture

 Color difference (L value)

 -18 ° C 37 ° C Control

 Detergent without DNase and without protease

     84.7

 Detergent and DNase and without protease

     90.3

 Detergent and DNase and without protease

 89.5 89.2

 Detergent and DNase and protease 1 and 2 g / l of protease inhibitor system 1

 91.0 90.7

 Detergent and DNase and protease 1 and 4 g / l of protease inhibitor system 1

 89.5 88.7

 Detergent and DNase and protease 1 and 2 g / l of protease inhibitor system 2

 89.7 90.3

 Detergent and DNase and protease 2 and 2 g / l of protease inhibitor system 1

 91.5 90.8

 Detergent and DNase and protease 2 and 2 g / l of protease inhibitor system 2

 90.3 89.7

Example 7

Added effect of DNase

[0502] To investigate whether the observed effects of deep cleaning and bleaching of the DNase of A. oryzae are unique to the DNase of A. oryzae in comparison to the existing laundry, a comparative study was performed. Brevundimonas sp. in round samples (2 cm diameter) of polyester (WFK30A) for 1 day (see example 4 - preparation of donor samples).

[0503] Five samples rinsed with Brevundimonas sp. they were mixed with five sterile samples in a 50 mL test tube and 10 mL of model A detergent wash solution (for composition, see example 4) containing 0.7 g / L of dirt (Pigmentschmutz, 09V, wfk, Krefeld, Germany) and enzyme. The following enzymes were used: Aspergillus oryzae DNase (0.04 ppm) (SEQ ID N °: 2), Celluclean ™ 5000L (0.002 ppm), Celluclean ™ 5.0T (0.001 ppm), Carezyme ™ (0.2 ppm) , Savinase ™ (2.7 ppm), Lipex ™ (0.06 ppm) and Stainzyme ™ (0.2 ppm) (all enzymes supplied by Novozymes A / S). The concentration in ppm represents the enzyme concentration in the wash solution. In parallel, a control was made without added enzyme.

[0504] The test tubes were placed in a Stuart rotor (MiniLOM) for 1 hour at 30 ° C. The samples were rinsed twice with tap water and dried on filter paper overnight. The color difference (L value) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted. The present example shows that the DNase of A. oryzae prevents the deposition of dirt (anti-redeposition) and improves whitening as the only of the laundry enzymes evaluated, indicating that the effect of deep cleaning and bleaching by the DNase of A. Oryzae is an added effect compared to the existing laundry enzymes.

Table 4

 Enzyme

 Detergent

 Model A detergent Model T detergent

 DNase

 89.6 89.6

 Celluclean ™ 5000L

 83.6 -

 Celluclean ™ 5.0T

 - 83.9

 Carezyme ™

 83.2 83.1

 Savinase ™

 83.8 84.9

 Lipex ™

 82.4 84.4

 Stainzyme ™

 82.2 83.6

 Without enzyme

 82.6 84.3

Example 8

Sweat smell detection on running shirts

[0505] Two white running shirts made of 100% polyester were pre-washed in a washing machine using 3.33 g / L of Model A detergent.

[0506] The two shirts were worn by a test person (man), one shirt at a time. The test person wore each of the shirts during physical activity for one hour. After washing, a shirt was washed in a washing machine using 3.33 g / L of model A detergent with the DNase of SEQ ID No. 2 (1 ppm), while the second shirt was washed in a washing machine using 3.33 g / L of model A detergent without DNase (0 ppm).

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The shirts were washed as described in the full scale wash described above and 15 L of tap water was used for washing. Both shirts were worn during physical activity and then washed. This wash and wash cycle was repeated 10 times.

[0507] For the evaluation of the smell, a trained evaluator investigated the shirts before use for sweat odor (bad smell) by smelling the shirts. If sweat smell was detected, a mark (X) was added in the following table. The following observations show that washing with DNase for 10 washes prevents odor accumulation.

Table 5

 Use number

 1 2 3 4 5 6 7 8 9 10

 T-shirt washed without DNasa

 - - - - - x x x x x

 DNasa washed shirt

Example 9

[0508] This example shows that the removal of enzyme stains such as protease, amylase, lipase, mannanase, pectate lyase and cellulase in a wash is not diminished by the presence of DNase.

[0509] Stain removal was shown in the following spots (effective enzyme in parentheses): CS-10 butter fat (lipase), chocolate milk / soot CS-03 (protease), CS-28 colored rice starch (amylase), CS-73 pigment gum (mannanase), CS-54 oatmeal / chocolate flour (cellulase), all from Center For Testmaterials BV, The Netherlands, and fresh 013KC banana (lycta pectate) from Warwick Equest Ltd ., United Kingdom.

[0510] All stains were cut into 4x9 cm samples except the C-S-10 butter fat, which was cut into 5x5 cm samples and the 013KC stain that was a 05 cm circular spot in a 10x10 cm sample. Two samples of each stain were fixed to a dishcloth (100% cotton) with a stapler. A dishcloth was added with the stained samples attached to each machine together with 3 kg of ballast consisting of: 3 shirts (100% cotton), 10 short-sleeved shirts (55% cotton, 45% polyester), 4 pillowcases (35% cotton, 65% polyester) (110x75 cm), 1 small bed sheet (100% cotton) (100x75 cm), and 4 tea towels (100% cotton).

[0511] The washings were done in Miele Softtronic W2445 washing machines at 30 ° C using a standard washing program with 12-13 liters of water for 3 kg of textile. Washing was done with 3.33 g / L of model A detergent as described above in Bagsv ^ rd tap water and a HI 9635 conductivity meter from Hanna Instruments, Canada, was used to measure the conductivity of water at 919 jS at 21 ° C.

[0512] Enzymes were added to the 4 washes in the amounts described in the following table:

Table 6

 Machine

 1 2 3 4

 DNase (SEQ ID No.: 2)

 - 0.04 ppm - 0.04 ppm

 Protease (Liquanase 2.5 L)

 - - 874 jL 874 jL

 Amylase (Amplify 12 L)

 - - 151 jL 151 jL

 Lipase (Lipex 100 L)

 - - 98 jL 98 jL

 Mananasa (Mannaway 4 L)

 - - 100 jL 100 jL

 Lycta pectate (Xpect 1000 L)

 - - 50 jL 50 jL

 Cellulase (Celluclean 5000 L)

 - - 821 jL 821 jL

[0513] Enzymes were added to washing machines in separate plastic beakers dissolved in 100 mL of water with 15 ° dH (water with 15 ° dH: 3.00 mL of 0.713 mol / L of CaCI2, 1.50 mL of 0.357 mol / L of MgCl2 and 0.3371 g of NaHCO3 in a measuring cylinder of 1 L, filled 1 L with MilliQ water). A separate plastic beaker was used for DNase, another plastic beaker was used for protease and the remaining enzymes (amylase, lipase, mannanase, lycta pectate and cellulose) were dissolved in a third plastic beaker. . The beakers were placed inside the drum of the washer before washing began.

[0514] After washing, the samples were dried overnight on filter paper and the remission was measured at 460 nm as described above. Samples of C-S-10 butter fat were measured 22 hours after the end of washing.

[0515] The average remission at 460 nm for stained samples after washing is shown in the following table. The higher the remission value, the more clean the sample after washing.

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 Machine

 1 2 3 4

 Enzymes added:

 DNasa P, A, L, M, PL, C P, A, L, M, PL, C + DNase

 Remission at 460 nm

 Oatmeal / Chocolate

 40.35 9.64 57.22 58.30

 Fresh banana

 52.50 47.61 68.37 73.97

 Chocolate milk

 36.55 36.61 56.11 55.66

 Garrofín rubber

 42.63 44.05 55.11 54.65

 Rice starch

 51.57 47.09 69.17 73.97

 Butter fat

 52.48 52.06 56.44 56.58

 P = protease, A = amylase, L = lipase, M = mannanase, PL = pectate lyase, C = cellulase

[0516] Based on a bilateral T test (with uneven variance), the mean remission values for each type of stain in wash 3 are not statistically significantly different from wash 4 at a 5% level of importance. The results show that the removal of enzyme stains such as protease, amylase, lipase, mannanase, pectate lyase and cellulase is not diminished by the presence of DNase in a wash.

Example 10

[0517] A washing experiment was conducted on a collection of running clothes (sweatshirts and running shirts), which had been worn during training exercises for at least 50 times and washed in between. During training exercises, running clothes get wet with sweat, but they usually don't get dirty. The running clothes had been washed previously in a Miele Softtronic W5825 at 30 ° C (with low machine load) using BioTex Black (Unilever) at a recommended standard dose.

[0518] Before conducting the present experiment, the sweatshirt and running shirt (100% polyester) were evaluated by a trained odor panelist, who found a pronounced smell of sour sweat (bad smell), particularly present in the snuff of the garments. The bad smell was present after washing the clothes using BioTex Black and the bad smell accumulated with the number of times the running clothes were used.

[0519] The running clothes were washed in a Miele Softtronic W5825 at 30 ° C (with low machine load) using standard dose of BioTex Black and a dose of DNase of A. oryzae (0.4 ppm) in the solution washing

[0520] Between each of the training exercises, the running clothes underwent another evaluation cycle by the odor panelist. The odor panelist found that the bad smell was significantly reduced and was barely detectable after 1 to 3 wash cycles in the presence of the A. oryzae DNase of the present invention. In an extended study, the running clothes were washed in the presence of the A. oryzae DNase of the present invention each time the clothes were washed. The odor panelist discovered that when the running clothes had been washed in the presence of the DNase of A. oryzae, they could be worn for at least two training exercises (allowing the clothes to dry without washing the clothes between the exercises of training) before the bad smell was clearly detectable.

Example 11

Stability of Aspergillus oryzae DNase, comparative study Preparation of sample detergent

[0521] The sample detergent was prepared according to the specifications in the following table (table 8) and stored for four weeks at a constant temperature (-18 ° C, 30 ° C and 37 ° C, respectively).

Table 8

 Detergent name

 DNase in detergent Savinase in detergent DNase in the washing solution Savinase in the washing solution

 Model A detergent

 BI DNase: 120 ppm 0 ppm BI DNase: 0.4 ppm 0 ppm

 Model A detergent

 BI DNase: 120 ppm 440 ppm BI DNase: 0.4 ppm 1.5 ppm

 Model A detergent

 Ao DNase: 120 ppm 0 ppm Ao DNase: 0.4 ________________ 0 ppm

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 Model A detergent

 Ao DNase: 120 ppm 440 ppm Ao DNase: 0.4 ppm 1.5 ppm

 Ariel Actilift Color & Style

 BI DNase: 120 ppm ND BI DNase: 0.4 ppm ND

 Ariel Actilift Color & Style

 Ao DNase: 120 ppm ND Ao DNase: 0.4 ppm________________ ND

 BI DNase: Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11); DNase of Ao: DNase of Aspergillus oryzae; Savinase (protease, sequence set forth in SEQ ID NO: 10), ND: no information available.

Preparation of donor samples with biofilm

[0522] Brevundimonas sp. in tryptone soy agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, United Kingdom) for 2-5 days at 30 ° C. From a single colony, a complete loop was transferred to 10 mL of TSB and incubated for 1 day at 30 ° C with shaking (240 rpm). After propagation, Brevundimonas sp. by centrifugation (Sigma Laboratory Centrifuge 6K15) (3000 g at 21 ° C in 7 min) and resuspended in 10 mL of TSB diluted twice with water. The optical density (OD) at 600 nm was measured using a spectrophotometer (POLARstar Omega (BMG Labtech, Ortenberg, Germany) .The fresh TSB diluted twice with water was inoculated to an OD600nm of 0.03, and 20 mL was added to a Petri dish, where a 5 cm x 5 cm sample of sterile WFK30A polyester was placed After incubation (24 h at 15 ° C with shaking (100 rpm), the samples were rinsed twice with 0.9% (p / v) NaCl The round samples (1 cm in diameter) were subsequently drilled from the 5 cm x 5 cm donor samples. The round samples were used for the washing experiment.

Washed

[0523] The wash solution was prepared by weighing the amount of detergent needed for the test in a flask, ie, model A detergent 3.3 g / L; Ariel Actilift Color & Style 6.9 g / L dissolved in sterile MilliQ water with a hardness of 15 ° dH. The dirt was prepared by resuspend the dirt (Pigmentschmutz, 09V, wfk, Krefeld, Germany) in tap water and stirring the suspension for 30 min. before use. The dirt was subsequently added to each dirt flask to reach a concentration of 0.35 g of dirt / L (Pigmentschmutz, 09V, wfk, Krefeld, Germany) and the flasks were filled with tap water to the final volume (4 ml ).

[0524] Round samples were placed on a 24-well format plate (two round samples obtained from the same donor sample per well). A mechanical tension object (MSO), in the form of an elongated metal object (1 cm) covered with plastic, was placed in each well. The plate was placed in a Hamilton robot and underwent a washing simulation program using the following conditions: washing cycle duration: 30 minutes with stirring (1000 rpm); temperature 30 ° C; volume of wash solution (total): 4 ml per well. During the washing time, the plate is agitated orbitally to bring the test solution into contact with the textile and apply mechanical tension in a regular regular oscillating manner. After the completion of the wash simulation cycle, the samples were removed from the wash solution and allowed to dry on a filter paper. The dried samples were fixed on a white sheet of paper for scanning.

[0525] The washing performance is measured as the color brightness of the washed textile. The brightness can also be expressed as the intensity of the light reflected from the sample when it is illuminated with white light. When the sample is stained, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure the washing performance. The color measurements are made with a flatbed scanner (EPSON V750 PRO), which is used to capture an image of the washed textile. To extract a value for light intensity from the scanned images, 24-bit pixel values of the image are converted to values for red, green and blue (RGB). The intensity value (int) is calculated by adding the RGB values as vectors and then taking the length of the resulting vector:

Int = & r2 + g2 + b2

Table 9

 Detergent

 Enzyme (s) Intensity (-18 ° C) Intensity (30 ° C) Intensity (37 ° C) AI Intensity (-18 ° C - 30 ° C) AI Intensity (-18 ° C - 37 ° C)

 Model A detergent

 DNase of BI 439 439 438 <5 <5

 Model A detergent

 DNase of BI + Savinase 436 430 416 5 20

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 Model A detergent

 DNase of Ao 439 438 437 <5 <5

 Model A detergent

 DNase of Ao + Savinase 439 439 438 <5 <5

 Ariel Actilift Color & Style

 DNase of BI 439 438 439 65 26

 Ariel Actilift Color & Style

 DNase of Ao 439 438 439 <5 <5

 Model A detergent

 - 350

 Ariel Actilift Color & Style

 - 392

BI DNase: Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11); DNase of Ao: DNase of Aspergillus oryzae, Savinase (protease, sequence set forth in SEQ ID NO: 10) .______

[0526] Conclusions: This comparative study demonstrates that the Aspergillus oryzae DNase of the present invention has superior stability towards proteases compared to the Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11 ).

Example 12

[0527] Stability of the Aspergillus oryzae DNase and the Bacillus licheniformis DNase, a comparative study in the miniLOM trial.

[0528] To compare the storage stability of the Bacillus licheniformis DNase and the Aspergillus oryzae DNase of the present invention, the storage stability of these two types of DNase was configured in the A and Ariel Actilift Color & Style model detergent. Detergent samples were prepared as described in example 11 and stored for two and four weeks at a constant temperature (-18 ° C, 35 ° C). The dosage of DNase in the wash solution was 0.4 ppm. The protease dosage (Savinase, sequence set forth in SEQ ID No. 10)) in the wash solution was 0.15 ppm. After storage, wash performance was evaluated in the miniLOM assay (described herein) using biofilm samples from Brevundimonas sp. of polyester (prepared as described in example 11). The washing performance is measured as the brightness of the color of the washed textile. The color difference (L values) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted.

Table 10

 Detergent

 Incubation time Enzymes L (-18 ° C) L (35 ° C) AL

 Model A detergent

 2 weeks DNase of BI 90.6 90.5 <0.5

 Model A detergent

 2 weeks DNase of BI + Savinase 91.5 84.6 6.9

 Model A detergent

 2 weeks DNase of Ao 90.4 90.5 <0.5

 Model A detergent

 2 weeks DNase of Ao + Savinase 90.5 91.1 <0.5

[0529] The recently prepared model A detergent without enzymes gave a color difference (L) of 82.6. Color difference (L), delta color difference (AL). BI DNase: Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11); DNase of Ao: DNase of Aspergillus oryzae.

Table 11

 Detergent

 Incubation time Enzymes L (-18 ° C) L (35 ° C) AL

 Ariel Actilift Color & Style

 2 weeks DNase of BI 92.3 87.0 5.3

 Ariel Actilift Color & Style

 2 weeks DNase of Ao 92.1 92.4 <0.5

 Detergent

 Incubation time Enzymes L (-18 ° C) L (37 ° C) AL

 Ariel Actilift Color & Style

 4 weeks DNase of BI 93.2 87.0 6.2

 Ariel Actilift Color & Style

 4 weeks DNase of Ao 93.2 93.0 <0.5

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[0530] The freshly prepared Model A detergent without enzymes gave a color difference (L) of 86.9. Color difference (L), delta color difference (AL). BI DNase: Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11); DNase of Ao: DNase of Aspergillus oryzae

[0531] Conclusions: This comparative study demonstrates that the Aspergillus oryzae DNase has superior stability towards proteases compared to the Bacillus licheniformis DNase (the mature peptide of the sequence set forth in SEQ ID No. 11).

Example 13

Odor removal by DNase (electronic nose)

Preparation of DNA samples with E-2-nonenal

[0532] Chromosomal DNA was isolated from Pseudomonas sp. using QlAamp DNA Blood Mini Kit following the manufacturer's instructions (Qiagen, Hilden, Germany). The polyester samples (WFK30A) (2 cm in diameter) were sterilized in a Holm & Halby Systec DB-23 autoclave for 60 minutes at 121 ° C. 100 | _il of Pseudomonas sp. DNA was added. purified diluted to a final concentration of 1 ng / | _il in each of the autoclaved samples and the samples were dried under continuous flow in a laminar air flow bank for 2 hours. After drying, 10 DNA samples were placed in a sterile 25 mL NUNC tube (364238; Thermo Scientific), and 10 ml of 0.2 mM E-2-nonenal (255653; Sigma-Aldrich) was added. The tube was placed in a Stuart rotor (20 rpm at room temperature for 20 min). The 10 samples were transferred to a 50,000 MWCO centrifuge tube (VS203, Vivaspin 20, Satorius). The tubes were centrifuged at 3000 g at 21 ° C in 1 min, and the 10 samples were divided into 2 sterile NUNC tubes (364238; Thermo Scientific) with 5 samples in each tube. Additionally, 5 samples of sterile polyester (WFK30A) (2 cm in diameter) without DNA and E-2-nonenal were added to each of the tubes. Samples without DNA were labeled to allow differentiation from samples with DNA / E-2-nonenal.

Procedure for washing DNA samples with E-2-nonenal

[0533] Washing solutions of Ariel Actilift Color & Style powder and Ariel Actilift powder were prepared by dissolving 5.0 g of detergent in 1000 ml of sterile MilliQ water with a hardness of 15 ° dH (EU conditions). A wash solution of Tide Pods was prepared by dissolving 1.8 g of the white phase detergent in 1000 ml of sterile milliQ water with a hardness of 6 ° dH. The wash solutions were left on a magnetic stirrer for 20 min before use.

[0534] The wash solution (10 ml) was added to each of the two identical tubes with 5 DNA samples with E-2-nonenal and 5 sterile samples, 10 _il of Aspergillus oryzae DNase was added herein invention to one of the tubes resulting in a final concentration of 5 ppm. The tubes were placed in a Stuart rotator (20 rpm at 30 ° C for 60 min). The wash solution was poured out, and the samples were rinsed twice with 20 mL of sterile milliQ water with hardness 15 ° dH. Samples from each tube were transferred to a 50,000 MWCO centrifuge tube (VS203, Vivaspin 20, Satorius) and centrifuged at 3000 g at 21 ° C in 1 min. Each sample with E-2- nonenal was then transferred to a 20 mL GC empty space bottle, using sterile clean tweezers to place each of the samples in each bottle. They were then analyzed on an electronic nose of Flash MOS HERACLES Flash gas chromatography, equipped with a Restek MXT-5 capillary column, with an HS100 autosampler and an FID detector.

Table 12. Intensity of E-2-nonenal measured with electronic nose.

 Detergent

 Maximum value area (without DNase) Maximum value area (with DNase)% reduction

 Ariel Actilift Powder

 108325 83177 23%

 Ariel Actilift Color & Style powder

 167288 122868 27%

 Tide pods

 128945 80895 37%

[0535] Conclusion: the odor retained in DNA in textiles can be removed with the Aspergillus oryzae DNase, resulting in a reduction in odor.

Example 14

Odor removal by DNase (sensory analysis)

[0536] Polyester and cotton samples (2 cm in diameter) were soiled with DNA from Pseudomonas sp. and 0.5 mM of E-2-nonenal (255653; Aldrich Sigma) (see example 13 for details of sample preparation of

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DNA) and washed in a Stuart rotator for 60 min at 30 ° C in model A detergent (prepared by dissolving 3.33 g in 1 liter of milliQ water with 15 ° dH hardness) in the presence or absence of Aspergillus oryzae DNase the present invention (5 ppm). After washing, the samples were rinsed twice in 20 ml of MilliQ water with a hardness of 15 ° dH. The samples were evaluated for the perceived intensity of E-2-nonenal by four panelists of formed odors (where the samples were blind). For the evaluation a scale of perceived intensity of 0 to 9 was used. 0 indicated no perceived intensity of E-2-nonenal, while 9 indicated the highest perceived intensity of E-2-nonenal. The results showed consensus among the four odor panelists.

Table 13

 Type of textile

 Addition of DNasa Average score of the four odor panelists

 Cotton

 - 5.0

 Cotton

 + 1.3

 Polyester

 - 6.3

 Polyester

 + 2.8

[0537] The odor retained in the DNA in the textile can be eliminated with the DNase of Aspergillus oryzae resulting in a reduction in odor.

Example 15

Test for visual determination of odor elimination

[0538] Brevundimonas sp. in tryptone soy agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, United Kingdom) for 2-5 days at 30 ° C. From a single colony, a complete loop was transferred to 10 mL of TSB and incubated for 20 hours at 30 ° C with shaking (240 rpm). After propagation, Brevundimonas sp. by centrifugation (Sigma Laboratory Centrifuge 6K15) (3000 g at 21 ° C in 7 min) and resuspended in 10 mL of TSB diluted twice with water. The optical density (OD) at 600 nm was measured using a spectrophotometer (POLARstar Omega (BMG Labtech, Ortenberg, Germany) .The fresh TSB diluted twice with water was inoculated for an OD600nm of 0.03, and 20 mL was added to a Petri dish, where a 5 cm x 5 cm sample of sterile WFK30A polyester was placed After incubation (24 h at 15 ° C with shaking (75 rpm), the samples were rinsed twice with 0.9% ( p / v) NaCl The samples were washed in TOM either directly after rinsing or after drying for 24 hours in a LAF bank.

[0539] The phenol red visual indicator agent in the solution was prepared by dissolving 0.1 g in 100 ml of 99% ethanol. Indicator solution (50 | _iL) was added to a filter paper (2 cm diameter) and dried overnight in the extractor hood to form an odor capture agent that includes the visual indicator agent (phenol red ). Four washed samples were placed at the bottom of a glass container (one container for each sample) and 500 pL of 17% TSB was added. As a control, the washed clean textile polyester was placed at the bottom of another glass container and 500 pL of 17% TSB was added. The containers with the samples were incubated at 37 ° C. The containers were monitored for up to 48 hours to detect color change of the red phenol tracer.

Table 14. Incubation results

 Detergent

 Aspergillus oryzae DNase enzyme (10 ppm)

 Original Tide

 Red (24 h) Yellow (24 h)

 Ariel sensitive White & Color

 Red (48 h) Yellow (48 h)

 Model T detergent

 Red (24 h) Yellow (24 h)

 The hours in the table indicate time points for color change detected.

Example 16

Effect of the presence of DNase in the detergent on the biofilm of Pseudomonas aeruginosa in textile

[0540] A strain of Pseudomonas aeruginosa isolated from the laundry was used in the present example. The P. aeruginosa strain was precultured in tryptone soy agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, United Kingdom) for 3 days at 30 ° C. From a single colony, a complete loop was transferred to 10 mL of TSB and incubated overnight at 30 ° C with shaking (200 r.p.m.). After propagation, overnight culture was diluted 1000 times in fresh TSB medium and 1.62 mL of this culture dilution was added in each well of a 12-well flat bottom polystyrene microplate (3512; Corning Incorporated, Corning, NY, USA), where round samples (2 cm diameter) of sterile polyester (WFK30A) were placed. After 48 h at 30 ° C with stirring (70 rpm), the culture medium was removed, and the samples were rinsed twice with 3 mL of 0.9% NaCl (w / v).

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[0541] Five samples rinsed with P. aeruginosa were placed in a 50 mL test specimen and 10 mL of model A liquid detergent prepared in water with a hardness of 15 ° dH with 0.7 g / L pigment dirt was added (Pigmentschmutz 09V, wfk, Krefeld, Germany) and the Aspergillus oryzae DNase of the present invention (0.1 ppm in the wash solution). As a control, a wash without DNase was performed in parallel. Washing was performed on a Stuart rotator for 1 hour at 30 ° C (test with miniLOM as described herein). The wash solution was removed, and the samples were rinsed twice with tap water and dried on filter paper overnight.

[0542] The color difference (L value in the L * a * b color space) was measured using a Minolta CR-300 chromometer head and a Minolta DP-301 data processor. The color difference (value L, L *) represents the darkest black at L * = 0, and the brightest white at L * = 100.

[0543] The results show that DNase is able to reduce stickiness and / or reduce the amount of P. aeruginosa biofilm in the textile.

Table 15. Effect of DNase on detergent in the biofilm of Pseudomonas aeruginosa in textile.

 DNase concentration (ppm)

 L value L value (with DNase) - L value (without DNase)

 0

 84.6

 0.1

 87.3 2.7

Example 17

DNase washing performance in the presence of optical brightener

Materials and methods

Preparation of biofilm samples

[0544] Round samples (2 cm diameter) of WFK30A sterile polyester with Brevundimonas sp. as described in example 4.

Washing experiment

[0545] Powder model T detergent washing solution without bleach was prepared by dissolving detergent (5.33 g / l) in water with a hardness of 15 ° dH. The AEO Biosoft N25-7 (NI) component (0.16 g / l) of the Model T detergent was added separately. Pigment dirt (Pigmentschmutz, 09V, wfk, Krefeld, Germany) (0.7 g / L) was added to the wash solution. The wash solution (10 ml) was added to a 50 ml tube with five samples rinsed with Brevundimonas sp. and five samples of sterile polyester (WFK30A). In the washings where DNase was included, DNase (0.5 ppm) was added. In the washings where optical brightener was included, Tinopal CBS-X (0.1 g / l) was added to the wash solution. The washing was carried out in a Stuart rotator for 1 hour at 30 ° C (test with miniLOM as described in this document). After washing, the samples with Brevundimonas sp. they were rinsed twice in tap water and dried on filter paper overnight. The color difference (L values) was measured using a Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer). The measurements were made without UV in the incident light and the L value of the CIE Lab color space was extracted.

Table 16. DNase wash performance in the presence of an optical brightener.

 Detergent model T (g / l) + NI (g / l)

 CBS-X (g / l) DNase of A. oryzae (ppm) L value Delta L value (L value - L value (no optical brightener, no DNase)

 5.33 + 0.16

 0 0 85.4

 5.33 + 0.16

 0 0.5 89.3 3.9

 5.33 + 0.16

 0.1 0 83.9 <0

 5.33 + 0.16

 0.1 0.5 89.3 0

[0546] The data presented in Table 16 demonstrate that the washing performance of the A. oryzae DNase of the present invention is not affected by the presence of an optical brightener.

Sequence listing

[0547]

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<110> Novozymes A / S <120> Detergent composition <130> 12785-WO-PCT <160> 12

<170> PatentIn version 3.5

<210> 1 <211> 910 <212> DNA

<213> Aspergillus oryzae

<220>

<221> exon <222> (1) .. (242)

<220>

<221> Intron <222> (243) .. (308)

<220>

<221> exon <222> (309) .. (494)

<220>

<221> Intron <222> (495) .. (555)

<220>

<221> exon <222> (556) .. (714)

<220>

<221> Intron <222> (715) .. (765)

<220>

<221> exon <222> (766) .. (907)

<220>

<221> Intron <222> (908) .. (910)

<400> 1

atg cag ctt act aag tcc etc ctg Met Gln Leu Thr Lys Ser Leu Leu 1 5

act cag falls gtt cta gct gtg cct Thr Gln His Val Leu Ala Val Pro 20

age gtc gaa aat gtt gee ctt aaa Ser Val Glu Asn Val Ala Leu Lys 35 40

 gta

 ttc gcg ctt tac atg ttt ggc

 Val

 Phe 10 Ala Leu Tyr Met Phe 15 Gly

 gtc

 aat ccc gag cct gat gct acg

 Val 25

 Asn Pro Glu Pro Asp 30 Ala Thr

 here

 ggc age ggt gat age cag age

 Thr

 Gly Ser Gly Asp 45 Ser Gln Ser

48

96

Claims (11)

5 10 fifteen twenty 25 30 35 40 1. Use of a polypeptide that has DNase activity to prevent, reduce or eliminate a biofilm of an article, where the polypeptide is obtained from a fungal source and the article is a textile and where the polypeptide that has DNase activity has at least 80% sequence identity with the mature polypeptide of SEQ iD #: 9. 2. Use according to claim 1 to prevent, reduce or eliminate the stickiness of the article. 3. Use according to any of claims 1 or 2 to pretreat the stains in the article. 4. Use according to any of claims 1-3 to prevent, reduce or eliminate redeposition of the dirt during a wash cycle. 5. Use according to any of claims 1-4 to prevent, reduce or eliminate adhesion of dirt to the article. 6. Use according to any of the preceding claims to maintain or improve the whiteness of the article. 7. Use according to any of the preceding claims, wherein the bad smell is reduced or eliminated from the article. 8. Detergent composition comprising a polypeptide having deoxyribonuclease activity (DNase) and a detergent adjunct ingredient, where the polypeptide is obtained from a fungal source and, where the polypeptide is obtained from Aspergillus. 9. Detergent composition according to claim 8, wherein the detergent attachment ingredient is selected from the group consisting of surfactants, adjuvants, flocculation aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleaching activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay dirt removal / antiredeposition agents, brighteners, soaps suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners fabrics, carriers, hydrotropes, adjuvants and adjuvants, tissue matting agents, antifoaming agents, dispersants, processing aids, and / or pigments. 10. Detergent composition according to claim 8, wherein the attached ingredient is a protease. 11. Washing method for washing an article that includes the steps of: to. Exposing an article to a wash solution comprising a polypeptide where the polypeptide having DNase activity has at least 80% sequence identity with the mature polypeptide of SEQ ID No. 9 or a detergent composition according to any of claims 8-10; b. Complete at least one wash cycle; Y C. Optionally rinse the item, Where the article is a textile.